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eNitric Oxide-Cold Stress Signalling Crosstalk-Evolution of a Novel Regulatory

Mechanism

Ankita Sehrawat1 Ravi Gupta

1 and Renu Deswal

Molecular Plant Physiology and Proteomics Laboratory Department of Botany University of

Delhi Delhi-110007 India

Correspondence Dr Renu Deswal Plant Molecular Physiology and Proteomics

Laboratory Department of Botany University of Delhi Delhi-110007 India

E-mail rdeswalbotanyduacin

1-Both the authors have contributed equally

Received Date 28-Sep-2012 Revised Date 15-Jan-2013 Accepted Date 31-Jan-2013

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting typesetting pagination and proofreading process which may lead to

differences between this version and the Version of Record Please cite this article as doi

101002pmic201200445

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eKeywords Proteomics Cold stress responsive proteins S-nitrosylation Nitration S-

glutathionylation

Abbreviations Nitric Oxide NO Reactive oxygen species ROS Reactive nitrogen

species RNS Post-translational modifications PTMs Cold acclimation CA

Transcription factors TFs CRTDRE-binding factors CBFsDREBs S-

nitrosoglutathione GSNO Ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO

Abstract

Plants enhance their cold stress tolerance by cold acclimation a process which results in vast

reprogramming of transcriptome proteome and metabolome Evidences suggest NO

production during cold stress which regulates genes (especially the CBF cold stress signalling

pathway) diverse proteins including transcription factors and phosphosphingolipids About

59 (redox) 50 (defencestress) and 30 (signalling) cold responsive proteins are

modulated by NO based post translational modifications (PTMs) namely S-nitrosylation

tyrosine nitration and S-glutathionylation suggesting a cross-talk between NO and cold

Analysis of cold stress responsive deep proteome in apoplast mitochondria chloroplast and

nucleus suggested continuation of this cross-talk in sub-cellular systems Modulation of cold

responsive proteins by these PTMs right from cytoskeletal elements in plasma membrane to

transcription factors in nucleus suggest a novel regulation of cold stress signalling NO

mediated altered protein transport in nucleus seems an important stress regulatory

mechanism This review addresses the NO and cold stress signalling cross-talk to present the

overview of this novel regulatory mechanism

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e1 Introduction

Cold stress is one of the major threats to plantrsquos growth productivity and distribution Cold

stress causes frost damage to crops and soft fruits resulting in significant yield losses More

than half (52) of the worldrsquos land area is affected by cold stress [1] and this threat is

progressively increasing and becoming severe due to increasing greenhouse gases leading to

temperature extremes These greenhouse gases would triple the possibility of extreme cold in

Europe and north Asia making these areas more susceptible to cold induced crop damage [2]

Plants being poikilotherms cannot maintain a constant temperature during cold leading to

reduction in the rate of metabolic reactions Cold stress becomes severe when temperature

drops below zero degrees resulting in the formation of extracellular ice This further leads to

mechanical dehydration and osmotic stress Some plants have an ability to increase their

freezing tolerance when exposed to low but above zero temperatures by a process known as

cold acclimation [3] Enhanced cold tolerance during cold acclimation is the outcome of the

significant changes in the gene expression Microarray based experiments showed that 4-20

of the Arabidopsis genome is cold responsive [4] Cold stress signalling is mediated by ABA-

independent as well as ABA-dependent signalling pathways CBFsDREBs (C-repeat binding

factors dehydration responsive element binding) controls ABA-independent expression of

COR (Cold-regulated) genes while AREBABF (ABA-responsive element-binding

proteinABA-binding factor) and MYBMYC (myelocytomatosis oncogene myeloblastosis

oncogene) transcription factors (TFs) regulate the ABA-dependent signalling [3]

This vast re-programming of the transcriptome leads to several other changes including

alteration in membrane lipid composition reorganization of cytoskeletal elements increased

antioxidant activity accumulation of osmolytes (like sugars and amino acids) and activation

of many signaling components Recently evolution of nitric oxide (NO) was shown as a key

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eevent in abiotic stress conditions including cold NO is a gaseous signaling molecule involved

in an array of physiological processes [5] Being very reactive NO has a very short life of

only few seconds It reacts with diverse molecules to produce reactive nitrogen species

(RNS) For example it reacts with glutathione (GSH a low molecular weight thiol) to form

S-nitrosoglutathione (GSNO) and with superoxide to form peroxynitrite These RNS in turn

modify the proteins by NO based post-translational modifications (PTMs) namely S-

nitrosylation S-glutathionylation and tyrosine nitration As some of the proteins are TFs

their modulation by NO also affects gene expression during cold

Unfortunately there are very few studies describing the regulation of genes and proteins by

NO in cold stress [6 7 8 9] Furthermore till date there is no comprehensive attempt to

relate NO and cold stress signalling This review presents evidences of cold induced NO

evolution Modulation of the cold inducible genes and TFs provided initial clues of NO and

cold stress cross-talk This was further reinforced by studies pertaining to NO modulated

PTMs of cold responsive proteins In addition the role of NO in modulation of lipids is also

detailed to emphasize its multifacet regulation strengthening the existence of NO and cold

stress signalling cross-talk

2 Cold stress mediated nitric oxide evolution enriches nitric oxide storage pool

NO is produced by both oxidative and reductive pathways in plants [10] Oxidative pathway

of NO production includes oxidation of L-arginine polyamine and hydroxylamine Nitric

oxide Synthase (NOS)-like enzyme is a major enzyme involved in its oxidative production It

catalyzes the conversion of L-Arginine to citrulline and NO is a by product of the reaction

NOS-like activity showed 6 fold increase by cold in Pisum sativum leaves [8] 781 (at 4 ordmC

stress) and 1395 (at 0 ordmC stress) increase in Chorispora bungeana suspension cultures [11]

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eand 20 increase in the Brassica juncea seedlings [12] These results were further supported

when application of a NOS inhibitor N (omega)-nitro-l-arginine (L-NNA) resulted in 33

23 and 70 reduction in the NO production after 10 20 and 30 days cold stress

respectively in Solanum lycopersicum fruits [13]

Polyamines mediated NO production in Capsicumm annum leaves was inhibited by the

application of α-difluoromethylornithine (DFMO a polyamine biosynthesis inhibitor) leading

to 9 reduction in the NO evolution during cold [9] However hydroxylamine mediated NO

production in cold stress is not yet reported

The reductive pathways of the NO production are majorly mediated by Nitrate Reductase

(NR) NR can utilize both nitrate and nitrite as substrate however NO is generated only by

reduction of nitrite in the presence of NAD(P)H Cold stress treated Arabidopsis leaves

showed ~2 fold increase in nitrite level [14] and 3 fold increase in the NR activity [15]

Application of tungstate (1 mM a NR inhibitor) reduced 68 of the cold induced NO

production [14] supporting NR mediated NO production during cold stress

Besides NR root specific plasma membrane-bound nitriteNO reductase (NiNOR) and

xanthine oxidoreductase (XOR) also contribute to NO formation by reductive pathways

However any contribution of these two enzymes in cold is not reported till date To provide

an overview cold induced NO production in plants in different cold stress conditions (0-8 degC

and 1 h to 30 d) is summarized in Supplementary Table 1

Downstream effects of this NO accumulation included its involvement in Reactive oxygen

species (ROS) detoxification ROS are toxic and cause proteins carbohydrates lipids and

DNA damage which ultimately results in cell death Plants have efficient antioxidant

machinery including the ascorbate-glutathione cycle to protect against ROS induced

oxidative damage This cycle detoxifies hydrogen peroxide (H2O2) by utilizing antioxidant

(ascorbate and GSH) and the enzymes catabolizing these antioxidants such as ascorbate

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eperoxidase (APX) monodehydroascorbate reductase (MDHAR) glutathione reductase (GR)

and dehydroascorbate reductase (DHAR) Cold stress significantly increases the activities of

all the enzymes of ascorbate-glutathione cycle while NO scavenger (2-(4-Carboxyphenyl)-

4455-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and tungstate decreased their activity

in cold treated B ramiflora seeds [16] Besides increasing the enzymatic activities NO also

promote the production of the substrates (ascorbate and GSH) of ascorbate-glutathione cycle

in cold stress S-nitroso-N-acetylpenicillamine (SNAP a NO donor) increased GSH

accumulation while cPTIO and tungstate showed the reversal [16] Similarly Sodium

Nitroprusside (SNP a NO donor) increased ascorbate while cPTIO and L-NAME decreased

its content [11] These results clearly showed the role of NO in positively regulating

ascorbate-glutathione cycle in cold stress Activity of catalase (CAT involved in the

conversion of H2O2 into H2O and O2) is increased by SNP in cold [11] Decreased H2O2

content further confirmed the role of NO in H2O2 removal in cold [11] SNP activated

Superoxide dismutase (SOD remove superoxide radicals) also reaffirmed the role of NO in

ROS removal in cold stress

All these studies convincingly showed that cold induced NO helps in combating cold stress-

induced oxidative damage by increasing the activities of antioxidant enzymes leading to

accumulation of antioxidants which in turn decrease ROS levels (H2O2 and superoxide

content) NO also helps in maintaining the proline level Proline acts as osmolyte stabilizes

the cell membranes and scavange free radicles and thereby contributes to the cold tolerance

Application of tungstate L-NNA and cPTIO led to lesser cold induced proline accumulation

in Arabidopsis leaves [15] and Camelia sinsnesis pollen tubes [17] Also nia1nia2 a NR null

mutant of Arabidopsis was less freezing tolerant reiterating significance of NR generated NO

in cold tolerance due to lesser accumulation of proline Endogenous level of proline is

maintained by P5CS1 (proline synthase) and ProDH (proline dehydrogenase) two key

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eenzymes involved in Pro biosynthesis and degradation respectively Interestingly the

expression of P5CS1 was lower while the expression of ProDH was higher in the mutants

[15] In addition Viagra (an inhibitor of cyclic guanosine monophosphate (cGMP) specific

phosphodiesterase used to stimulate cGMP level) promoted proline accumulation and the

effect was partially reversed with L-NNA or cPTIO in cold [17] Therefore suggesting the

role of cGMP as a downstream target of NO mediated proline accumulation in cold stress

NO also contributes to S-nitrosothiols (SNOs) and GSNO formation which are the

endogenous reservoir of NO [18] Cold stress enhanced SNO accumulation up to 12 fold (4

degC 6 h) in B juncea seedlings [7] 5 fold (48 h 8 ordmC) in P sativum leaves [8] and 2 fold in B

ramiflora embryos [16] Confocal laser scanning microscopy (CLSM) further confirmed

SNO accumulation in vascular tissue palisade and spongy mesophyll after cold stress (1 d 8

degC) in C annum leaves [9] The effect of cold stress on GSNO content is not investigated yet

but increased activity of GSNO reductase (GSNOR) was observed in pepper leaves in cold

[9] indicating GSNO accumulation GSNOR catalyzes the reduction of GSNO to glutathione

disulfide (GSSH) and ammonia (NH3) GSNOR provides cellular protection against

nitrosative stress in cold stress by maintaining low endogenous levels of GSNO [19]

It is well established that cold stress induced NO production either by oxidative or reductive

pathways contributes to cellular storage pool in the form of GSNO and SNO which in turn act

as a depot of NO for its sustained release and reaction with varied types of enzymes including

antioxidant enzymes Besides it also modulates genes thus playing an important role in

modulating the cold stress responsive transcriptome

3 Transcriptional and posttranslational modulation of cold responsive transcriptome

and Transcription factors

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ePlants response to cold stress involves massive regulation of cold responsive genes such as

COR (COR COR78RD29A COR47 COR15a COR66) KIN (cold-induced) LTI (low-

temperature induced) RD (responsive to dehydration) Dehydrin (DHN) Late

EmbryogenesisndashAbundant (LEA) and Responsive To Abscisic Acid (RAB) [3] The

expression of these cold responsive genes is controlled by TFs A set of genes controlled by a

TF is known as a regulon These regulons are regulated either in an ABA-independent or

ABA-dependent manner

311 ABA-independent cold signaling pathway

ABA-independent signalling pathway includes (1) the CBFDREB regulon and (2) the NAC

(NAM ATAF and CUC) and ZF-HD (zinc-finger homeodomain) regulon CBF regulon is

best characterized in cold stress while NAC and ZF-HD regulon is mostly analyzed in

drought and salinity stress [20]

CBFDREB1s bind to C-repeatdehydration-responsive elements (CRTDRE) present in the

promoter of the COR genes and activate transcription CBFs belong to the ethylene

responsive element binding proteinsAPETALA2 family Three cold-responsive CBFs

(CBF1DREB1b CBF2DREB1c and CBF3DREB1a) were identified in Arabidopsis [21]

CBF1 and CBF3 transcripts are positively regulated by NO whereas CBF2 expression

remained unaltered in Arabidopsis [14] In tomato out of the three CBFs (LeCBF1-3) only

LeCBF1 is cold induced Application of SNP increased LeCBF1 expression whereas

nitroarginine (NOS competitive inhibitor) reduced its expression indicating involvement of

NOS-like enzyme in enhancing the expression of LeCBF1 [13] Apart from CBFs Cold

Regulated 15a gene (COR15a) Low temperature induced gene 30 (LTI30) and Low

temperature induced gene 78 (LTI78) are positively regulated by NO [14] suggesting

regulation of both cold induced TFs as well as genes by NO

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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e1 Introduction

Cold stress is one of the major threats to plantrsquos growth productivity and distribution Cold

stress causes frost damage to crops and soft fruits resulting in significant yield losses More

than half (52) of the worldrsquos land area is affected by cold stress [1] and this threat is

progressively increasing and becoming severe due to increasing greenhouse gases leading to

temperature extremes These greenhouse gases would triple the possibility of extreme cold in

Europe and north Asia making these areas more susceptible to cold induced crop damage [2]

Plants being poikilotherms cannot maintain a constant temperature during cold leading to

reduction in the rate of metabolic reactions Cold stress becomes severe when temperature

drops below zero degrees resulting in the formation of extracellular ice This further leads to

mechanical dehydration and osmotic stress Some plants have an ability to increase their

freezing tolerance when exposed to low but above zero temperatures by a process known as

cold acclimation [3] Enhanced cold tolerance during cold acclimation is the outcome of the

significant changes in the gene expression Microarray based experiments showed that 4-20

of the Arabidopsis genome is cold responsive [4] Cold stress signalling is mediated by ABA-

independent as well as ABA-dependent signalling pathways CBFsDREBs (C-repeat binding

factors dehydration responsive element binding) controls ABA-independent expression of

COR (Cold-regulated) genes while AREBABF (ABA-responsive element-binding

proteinABA-binding factor) and MYBMYC (myelocytomatosis oncogene myeloblastosis

oncogene) transcription factors (TFs) regulate the ABA-dependent signalling [3]

This vast re-programming of the transcriptome leads to several other changes including

alteration in membrane lipid composition reorganization of cytoskeletal elements increased

antioxidant activity accumulation of osmolytes (like sugars and amino acids) and activation

of many signaling components Recently evolution of nitric oxide (NO) was shown as a key

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eevent in abiotic stress conditions including cold NO is a gaseous signaling molecule involved

in an array of physiological processes [5] Being very reactive NO has a very short life of

only few seconds It reacts with diverse molecules to produce reactive nitrogen species

(RNS) For example it reacts with glutathione (GSH a low molecular weight thiol) to form

S-nitrosoglutathione (GSNO) and with superoxide to form peroxynitrite These RNS in turn

modify the proteins by NO based post-translational modifications (PTMs) namely S-

nitrosylation S-glutathionylation and tyrosine nitration As some of the proteins are TFs

their modulation by NO also affects gene expression during cold

Unfortunately there are very few studies describing the regulation of genes and proteins by

NO in cold stress [6 7 8 9] Furthermore till date there is no comprehensive attempt to

relate NO and cold stress signalling This review presents evidences of cold induced NO

evolution Modulation of the cold inducible genes and TFs provided initial clues of NO and

cold stress cross-talk This was further reinforced by studies pertaining to NO modulated

PTMs of cold responsive proteins In addition the role of NO in modulation of lipids is also

detailed to emphasize its multifacet regulation strengthening the existence of NO and cold

stress signalling cross-talk

2 Cold stress mediated nitric oxide evolution enriches nitric oxide storage pool

NO is produced by both oxidative and reductive pathways in plants [10] Oxidative pathway

of NO production includes oxidation of L-arginine polyamine and hydroxylamine Nitric

oxide Synthase (NOS)-like enzyme is a major enzyme involved in its oxidative production It

catalyzes the conversion of L-Arginine to citrulline and NO is a by product of the reaction

NOS-like activity showed 6 fold increase by cold in Pisum sativum leaves [8] 781 (at 4 ordmC

stress) and 1395 (at 0 ordmC stress) increase in Chorispora bungeana suspension cultures [11]

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eand 20 increase in the Brassica juncea seedlings [12] These results were further supported

when application of a NOS inhibitor N (omega)-nitro-l-arginine (L-NNA) resulted in 33

23 and 70 reduction in the NO production after 10 20 and 30 days cold stress

respectively in Solanum lycopersicum fruits [13]

Polyamines mediated NO production in Capsicumm annum leaves was inhibited by the

application of α-difluoromethylornithine (DFMO a polyamine biosynthesis inhibitor) leading

to 9 reduction in the NO evolution during cold [9] However hydroxylamine mediated NO

production in cold stress is not yet reported

The reductive pathways of the NO production are majorly mediated by Nitrate Reductase

(NR) NR can utilize both nitrate and nitrite as substrate however NO is generated only by

reduction of nitrite in the presence of NAD(P)H Cold stress treated Arabidopsis leaves

showed ~2 fold increase in nitrite level [14] and 3 fold increase in the NR activity [15]

Application of tungstate (1 mM a NR inhibitor) reduced 68 of the cold induced NO

production [14] supporting NR mediated NO production during cold stress

Besides NR root specific plasma membrane-bound nitriteNO reductase (NiNOR) and

xanthine oxidoreductase (XOR) also contribute to NO formation by reductive pathways

However any contribution of these two enzymes in cold is not reported till date To provide

an overview cold induced NO production in plants in different cold stress conditions (0-8 degC

and 1 h to 30 d) is summarized in Supplementary Table 1

Downstream effects of this NO accumulation included its involvement in Reactive oxygen

species (ROS) detoxification ROS are toxic and cause proteins carbohydrates lipids and

DNA damage which ultimately results in cell death Plants have efficient antioxidant

machinery including the ascorbate-glutathione cycle to protect against ROS induced

oxidative damage This cycle detoxifies hydrogen peroxide (H2O2) by utilizing antioxidant

(ascorbate and GSH) and the enzymes catabolizing these antioxidants such as ascorbate

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eperoxidase (APX) monodehydroascorbate reductase (MDHAR) glutathione reductase (GR)

and dehydroascorbate reductase (DHAR) Cold stress significantly increases the activities of

all the enzymes of ascorbate-glutathione cycle while NO scavenger (2-(4-Carboxyphenyl)-

4455-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and tungstate decreased their activity

in cold treated B ramiflora seeds [16] Besides increasing the enzymatic activities NO also

promote the production of the substrates (ascorbate and GSH) of ascorbate-glutathione cycle

in cold stress S-nitroso-N-acetylpenicillamine (SNAP a NO donor) increased GSH

accumulation while cPTIO and tungstate showed the reversal [16] Similarly Sodium

Nitroprusside (SNP a NO donor) increased ascorbate while cPTIO and L-NAME decreased

its content [11] These results clearly showed the role of NO in positively regulating

ascorbate-glutathione cycle in cold stress Activity of catalase (CAT involved in the

conversion of H2O2 into H2O and O2) is increased by SNP in cold [11] Decreased H2O2

content further confirmed the role of NO in H2O2 removal in cold [11] SNP activated

Superoxide dismutase (SOD remove superoxide radicals) also reaffirmed the role of NO in

ROS removal in cold stress

All these studies convincingly showed that cold induced NO helps in combating cold stress-

induced oxidative damage by increasing the activities of antioxidant enzymes leading to

accumulation of antioxidants which in turn decrease ROS levels (H2O2 and superoxide

content) NO also helps in maintaining the proline level Proline acts as osmolyte stabilizes

the cell membranes and scavange free radicles and thereby contributes to the cold tolerance

Application of tungstate L-NNA and cPTIO led to lesser cold induced proline accumulation

in Arabidopsis leaves [15] and Camelia sinsnesis pollen tubes [17] Also nia1nia2 a NR null

mutant of Arabidopsis was less freezing tolerant reiterating significance of NR generated NO

in cold tolerance due to lesser accumulation of proline Endogenous level of proline is

maintained by P5CS1 (proline synthase) and ProDH (proline dehydrogenase) two key

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eenzymes involved in Pro biosynthesis and degradation respectively Interestingly the

expression of P5CS1 was lower while the expression of ProDH was higher in the mutants

[15] In addition Viagra (an inhibitor of cyclic guanosine monophosphate (cGMP) specific

phosphodiesterase used to stimulate cGMP level) promoted proline accumulation and the

effect was partially reversed with L-NNA or cPTIO in cold [17] Therefore suggesting the

role of cGMP as a downstream target of NO mediated proline accumulation in cold stress

NO also contributes to S-nitrosothiols (SNOs) and GSNO formation which are the

endogenous reservoir of NO [18] Cold stress enhanced SNO accumulation up to 12 fold (4

degC 6 h) in B juncea seedlings [7] 5 fold (48 h 8 ordmC) in P sativum leaves [8] and 2 fold in B

ramiflora embryos [16] Confocal laser scanning microscopy (CLSM) further confirmed

SNO accumulation in vascular tissue palisade and spongy mesophyll after cold stress (1 d 8

degC) in C annum leaves [9] The effect of cold stress on GSNO content is not investigated yet

but increased activity of GSNO reductase (GSNOR) was observed in pepper leaves in cold

[9] indicating GSNO accumulation GSNOR catalyzes the reduction of GSNO to glutathione

disulfide (GSSH) and ammonia (NH3) GSNOR provides cellular protection against

nitrosative stress in cold stress by maintaining low endogenous levels of GSNO [19]

It is well established that cold stress induced NO production either by oxidative or reductive

pathways contributes to cellular storage pool in the form of GSNO and SNO which in turn act

as a depot of NO for its sustained release and reaction with varied types of enzymes including

antioxidant enzymes Besides it also modulates genes thus playing an important role in

modulating the cold stress responsive transcriptome

3 Transcriptional and posttranslational modulation of cold responsive transcriptome

and Transcription factors

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ePlants response to cold stress involves massive regulation of cold responsive genes such as

COR (COR COR78RD29A COR47 COR15a COR66) KIN (cold-induced) LTI (low-

temperature induced) RD (responsive to dehydration) Dehydrin (DHN) Late

EmbryogenesisndashAbundant (LEA) and Responsive To Abscisic Acid (RAB) [3] The

expression of these cold responsive genes is controlled by TFs A set of genes controlled by a

TF is known as a regulon These regulons are regulated either in an ABA-independent or

ABA-dependent manner

311 ABA-independent cold signaling pathway

ABA-independent signalling pathway includes (1) the CBFDREB regulon and (2) the NAC

(NAM ATAF and CUC) and ZF-HD (zinc-finger homeodomain) regulon CBF regulon is

best characterized in cold stress while NAC and ZF-HD regulon is mostly analyzed in

drought and salinity stress [20]

CBFDREB1s bind to C-repeatdehydration-responsive elements (CRTDRE) present in the

promoter of the COR genes and activate transcription CBFs belong to the ethylene

responsive element binding proteinsAPETALA2 family Three cold-responsive CBFs

(CBF1DREB1b CBF2DREB1c and CBF3DREB1a) were identified in Arabidopsis [21]

CBF1 and CBF3 transcripts are positively regulated by NO whereas CBF2 expression

remained unaltered in Arabidopsis [14] In tomato out of the three CBFs (LeCBF1-3) only

LeCBF1 is cold induced Application of SNP increased LeCBF1 expression whereas

nitroarginine (NOS competitive inhibitor) reduced its expression indicating involvement of

NOS-like enzyme in enhancing the expression of LeCBF1 [13] Apart from CBFs Cold

Regulated 15a gene (COR15a) Low temperature induced gene 30 (LTI30) and Low

temperature induced gene 78 (LTI78) are positively regulated by NO [14] suggesting

regulation of both cold induced TFs as well as genes by NO

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eevent in abiotic stress conditions including cold NO is a gaseous signaling molecule involved

in an array of physiological processes [5] Being very reactive NO has a very short life of

only few seconds It reacts with diverse molecules to produce reactive nitrogen species

(RNS) For example it reacts with glutathione (GSH a low molecular weight thiol) to form

S-nitrosoglutathione (GSNO) and with superoxide to form peroxynitrite These RNS in turn

modify the proteins by NO based post-translational modifications (PTMs) namely S-

nitrosylation S-glutathionylation and tyrosine nitration As some of the proteins are TFs

their modulation by NO also affects gene expression during cold

Unfortunately there are very few studies describing the regulation of genes and proteins by

NO in cold stress [6 7 8 9] Furthermore till date there is no comprehensive attempt to

relate NO and cold stress signalling This review presents evidences of cold induced NO

evolution Modulation of the cold inducible genes and TFs provided initial clues of NO and

cold stress cross-talk This was further reinforced by studies pertaining to NO modulated

PTMs of cold responsive proteins In addition the role of NO in modulation of lipids is also

detailed to emphasize its multifacet regulation strengthening the existence of NO and cold

stress signalling cross-talk

2 Cold stress mediated nitric oxide evolution enriches nitric oxide storage pool

NO is produced by both oxidative and reductive pathways in plants [10] Oxidative pathway

of NO production includes oxidation of L-arginine polyamine and hydroxylamine Nitric

oxide Synthase (NOS)-like enzyme is a major enzyme involved in its oxidative production It

catalyzes the conversion of L-Arginine to citrulline and NO is a by product of the reaction

NOS-like activity showed 6 fold increase by cold in Pisum sativum leaves [8] 781 (at 4 ordmC

stress) and 1395 (at 0 ordmC stress) increase in Chorispora bungeana suspension cultures [11]

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eand 20 increase in the Brassica juncea seedlings [12] These results were further supported

when application of a NOS inhibitor N (omega)-nitro-l-arginine (L-NNA) resulted in 33

23 and 70 reduction in the NO production after 10 20 and 30 days cold stress

respectively in Solanum lycopersicum fruits [13]

Polyamines mediated NO production in Capsicumm annum leaves was inhibited by the

application of α-difluoromethylornithine (DFMO a polyamine biosynthesis inhibitor) leading

to 9 reduction in the NO evolution during cold [9] However hydroxylamine mediated NO

production in cold stress is not yet reported

The reductive pathways of the NO production are majorly mediated by Nitrate Reductase

(NR) NR can utilize both nitrate and nitrite as substrate however NO is generated only by

reduction of nitrite in the presence of NAD(P)H Cold stress treated Arabidopsis leaves

showed ~2 fold increase in nitrite level [14] and 3 fold increase in the NR activity [15]

Application of tungstate (1 mM a NR inhibitor) reduced 68 of the cold induced NO

production [14] supporting NR mediated NO production during cold stress

Besides NR root specific plasma membrane-bound nitriteNO reductase (NiNOR) and

xanthine oxidoreductase (XOR) also contribute to NO formation by reductive pathways

However any contribution of these two enzymes in cold is not reported till date To provide

an overview cold induced NO production in plants in different cold stress conditions (0-8 degC

and 1 h to 30 d) is summarized in Supplementary Table 1

Downstream effects of this NO accumulation included its involvement in Reactive oxygen

species (ROS) detoxification ROS are toxic and cause proteins carbohydrates lipids and

DNA damage which ultimately results in cell death Plants have efficient antioxidant

machinery including the ascorbate-glutathione cycle to protect against ROS induced

oxidative damage This cycle detoxifies hydrogen peroxide (H2O2) by utilizing antioxidant

(ascorbate and GSH) and the enzymes catabolizing these antioxidants such as ascorbate

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eperoxidase (APX) monodehydroascorbate reductase (MDHAR) glutathione reductase (GR)

and dehydroascorbate reductase (DHAR) Cold stress significantly increases the activities of

all the enzymes of ascorbate-glutathione cycle while NO scavenger (2-(4-Carboxyphenyl)-

4455-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and tungstate decreased their activity

in cold treated B ramiflora seeds [16] Besides increasing the enzymatic activities NO also

promote the production of the substrates (ascorbate and GSH) of ascorbate-glutathione cycle

in cold stress S-nitroso-N-acetylpenicillamine (SNAP a NO donor) increased GSH

accumulation while cPTIO and tungstate showed the reversal [16] Similarly Sodium

Nitroprusside (SNP a NO donor) increased ascorbate while cPTIO and L-NAME decreased

its content [11] These results clearly showed the role of NO in positively regulating

ascorbate-glutathione cycle in cold stress Activity of catalase (CAT involved in the

conversion of H2O2 into H2O and O2) is increased by SNP in cold [11] Decreased H2O2

content further confirmed the role of NO in H2O2 removal in cold [11] SNP activated

Superoxide dismutase (SOD remove superoxide radicals) also reaffirmed the role of NO in

ROS removal in cold stress

All these studies convincingly showed that cold induced NO helps in combating cold stress-

induced oxidative damage by increasing the activities of antioxidant enzymes leading to

accumulation of antioxidants which in turn decrease ROS levels (H2O2 and superoxide

content) NO also helps in maintaining the proline level Proline acts as osmolyte stabilizes

the cell membranes and scavange free radicles and thereby contributes to the cold tolerance

Application of tungstate L-NNA and cPTIO led to lesser cold induced proline accumulation

in Arabidopsis leaves [15] and Camelia sinsnesis pollen tubes [17] Also nia1nia2 a NR null

mutant of Arabidopsis was less freezing tolerant reiterating significance of NR generated NO

in cold tolerance due to lesser accumulation of proline Endogenous level of proline is

maintained by P5CS1 (proline synthase) and ProDH (proline dehydrogenase) two key

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eenzymes involved in Pro biosynthesis and degradation respectively Interestingly the

expression of P5CS1 was lower while the expression of ProDH was higher in the mutants

[15] In addition Viagra (an inhibitor of cyclic guanosine monophosphate (cGMP) specific

phosphodiesterase used to stimulate cGMP level) promoted proline accumulation and the

effect was partially reversed with L-NNA or cPTIO in cold [17] Therefore suggesting the

role of cGMP as a downstream target of NO mediated proline accumulation in cold stress

NO also contributes to S-nitrosothiols (SNOs) and GSNO formation which are the

endogenous reservoir of NO [18] Cold stress enhanced SNO accumulation up to 12 fold (4

degC 6 h) in B juncea seedlings [7] 5 fold (48 h 8 ordmC) in P sativum leaves [8] and 2 fold in B

ramiflora embryos [16] Confocal laser scanning microscopy (CLSM) further confirmed

SNO accumulation in vascular tissue palisade and spongy mesophyll after cold stress (1 d 8

degC) in C annum leaves [9] The effect of cold stress on GSNO content is not investigated yet

but increased activity of GSNO reductase (GSNOR) was observed in pepper leaves in cold

[9] indicating GSNO accumulation GSNOR catalyzes the reduction of GSNO to glutathione

disulfide (GSSH) and ammonia (NH3) GSNOR provides cellular protection against

nitrosative stress in cold stress by maintaining low endogenous levels of GSNO [19]

It is well established that cold stress induced NO production either by oxidative or reductive

pathways contributes to cellular storage pool in the form of GSNO and SNO which in turn act

as a depot of NO for its sustained release and reaction with varied types of enzymes including

antioxidant enzymes Besides it also modulates genes thus playing an important role in

modulating the cold stress responsive transcriptome

3 Transcriptional and posttranslational modulation of cold responsive transcriptome

and Transcription factors

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ePlants response to cold stress involves massive regulation of cold responsive genes such as

COR (COR COR78RD29A COR47 COR15a COR66) KIN (cold-induced) LTI (low-

temperature induced) RD (responsive to dehydration) Dehydrin (DHN) Late

EmbryogenesisndashAbundant (LEA) and Responsive To Abscisic Acid (RAB) [3] The

expression of these cold responsive genes is controlled by TFs A set of genes controlled by a

TF is known as a regulon These regulons are regulated either in an ABA-independent or

ABA-dependent manner

311 ABA-independent cold signaling pathway

ABA-independent signalling pathway includes (1) the CBFDREB regulon and (2) the NAC

(NAM ATAF and CUC) and ZF-HD (zinc-finger homeodomain) regulon CBF regulon is

best characterized in cold stress while NAC and ZF-HD regulon is mostly analyzed in

drought and salinity stress [20]

CBFDREB1s bind to C-repeatdehydration-responsive elements (CRTDRE) present in the

promoter of the COR genes and activate transcription CBFs belong to the ethylene

responsive element binding proteinsAPETALA2 family Three cold-responsive CBFs

(CBF1DREB1b CBF2DREB1c and CBF3DREB1a) were identified in Arabidopsis [21]

CBF1 and CBF3 transcripts are positively regulated by NO whereas CBF2 expression

remained unaltered in Arabidopsis [14] In tomato out of the three CBFs (LeCBF1-3) only

LeCBF1 is cold induced Application of SNP increased LeCBF1 expression whereas

nitroarginine (NOS competitive inhibitor) reduced its expression indicating involvement of

NOS-like enzyme in enhancing the expression of LeCBF1 [13] Apart from CBFs Cold

Regulated 15a gene (COR15a) Low temperature induced gene 30 (LTI30) and Low

temperature induced gene 78 (LTI78) are positively regulated by NO [14] suggesting

regulation of both cold induced TFs as well as genes by NO

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eand 20 increase in the Brassica juncea seedlings [12] These results were further supported

when application of a NOS inhibitor N (omega)-nitro-l-arginine (L-NNA) resulted in 33

23 and 70 reduction in the NO production after 10 20 and 30 days cold stress

respectively in Solanum lycopersicum fruits [13]

Polyamines mediated NO production in Capsicumm annum leaves was inhibited by the

application of α-difluoromethylornithine (DFMO a polyamine biosynthesis inhibitor) leading

to 9 reduction in the NO evolution during cold [9] However hydroxylamine mediated NO

production in cold stress is not yet reported

The reductive pathways of the NO production are majorly mediated by Nitrate Reductase

(NR) NR can utilize both nitrate and nitrite as substrate however NO is generated only by

reduction of nitrite in the presence of NAD(P)H Cold stress treated Arabidopsis leaves

showed ~2 fold increase in nitrite level [14] and 3 fold increase in the NR activity [15]

Application of tungstate (1 mM a NR inhibitor) reduced 68 of the cold induced NO

production [14] supporting NR mediated NO production during cold stress

Besides NR root specific plasma membrane-bound nitriteNO reductase (NiNOR) and

xanthine oxidoreductase (XOR) also contribute to NO formation by reductive pathways

However any contribution of these two enzymes in cold is not reported till date To provide

an overview cold induced NO production in plants in different cold stress conditions (0-8 degC

and 1 h to 30 d) is summarized in Supplementary Table 1

Downstream effects of this NO accumulation included its involvement in Reactive oxygen

species (ROS) detoxification ROS are toxic and cause proteins carbohydrates lipids and

DNA damage which ultimately results in cell death Plants have efficient antioxidant

machinery including the ascorbate-glutathione cycle to protect against ROS induced

oxidative damage This cycle detoxifies hydrogen peroxide (H2O2) by utilizing antioxidant

(ascorbate and GSH) and the enzymes catabolizing these antioxidants such as ascorbate

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eperoxidase (APX) monodehydroascorbate reductase (MDHAR) glutathione reductase (GR)

and dehydroascorbate reductase (DHAR) Cold stress significantly increases the activities of

all the enzymes of ascorbate-glutathione cycle while NO scavenger (2-(4-Carboxyphenyl)-

4455-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and tungstate decreased their activity

in cold treated B ramiflora seeds [16] Besides increasing the enzymatic activities NO also

promote the production of the substrates (ascorbate and GSH) of ascorbate-glutathione cycle

in cold stress S-nitroso-N-acetylpenicillamine (SNAP a NO donor) increased GSH

accumulation while cPTIO and tungstate showed the reversal [16] Similarly Sodium

Nitroprusside (SNP a NO donor) increased ascorbate while cPTIO and L-NAME decreased

its content [11] These results clearly showed the role of NO in positively regulating

ascorbate-glutathione cycle in cold stress Activity of catalase (CAT involved in the

conversion of H2O2 into H2O and O2) is increased by SNP in cold [11] Decreased H2O2

content further confirmed the role of NO in H2O2 removal in cold [11] SNP activated

Superoxide dismutase (SOD remove superoxide radicals) also reaffirmed the role of NO in

ROS removal in cold stress

All these studies convincingly showed that cold induced NO helps in combating cold stress-

induced oxidative damage by increasing the activities of antioxidant enzymes leading to

accumulation of antioxidants which in turn decrease ROS levels (H2O2 and superoxide

content) NO also helps in maintaining the proline level Proline acts as osmolyte stabilizes

the cell membranes and scavange free radicles and thereby contributes to the cold tolerance

Application of tungstate L-NNA and cPTIO led to lesser cold induced proline accumulation

in Arabidopsis leaves [15] and Camelia sinsnesis pollen tubes [17] Also nia1nia2 a NR null

mutant of Arabidopsis was less freezing tolerant reiterating significance of NR generated NO

in cold tolerance due to lesser accumulation of proline Endogenous level of proline is

maintained by P5CS1 (proline synthase) and ProDH (proline dehydrogenase) two key

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eenzymes involved in Pro biosynthesis and degradation respectively Interestingly the

expression of P5CS1 was lower while the expression of ProDH was higher in the mutants

[15] In addition Viagra (an inhibitor of cyclic guanosine monophosphate (cGMP) specific

phosphodiesterase used to stimulate cGMP level) promoted proline accumulation and the

effect was partially reversed with L-NNA or cPTIO in cold [17] Therefore suggesting the

role of cGMP as a downstream target of NO mediated proline accumulation in cold stress

NO also contributes to S-nitrosothiols (SNOs) and GSNO formation which are the

endogenous reservoir of NO [18] Cold stress enhanced SNO accumulation up to 12 fold (4

degC 6 h) in B juncea seedlings [7] 5 fold (48 h 8 ordmC) in P sativum leaves [8] and 2 fold in B

ramiflora embryos [16] Confocal laser scanning microscopy (CLSM) further confirmed

SNO accumulation in vascular tissue palisade and spongy mesophyll after cold stress (1 d 8

degC) in C annum leaves [9] The effect of cold stress on GSNO content is not investigated yet

but increased activity of GSNO reductase (GSNOR) was observed in pepper leaves in cold

[9] indicating GSNO accumulation GSNOR catalyzes the reduction of GSNO to glutathione

disulfide (GSSH) and ammonia (NH3) GSNOR provides cellular protection against

nitrosative stress in cold stress by maintaining low endogenous levels of GSNO [19]

It is well established that cold stress induced NO production either by oxidative or reductive

pathways contributes to cellular storage pool in the form of GSNO and SNO which in turn act

as a depot of NO for its sustained release and reaction with varied types of enzymes including

antioxidant enzymes Besides it also modulates genes thus playing an important role in

modulating the cold stress responsive transcriptome

3 Transcriptional and posttranslational modulation of cold responsive transcriptome

and Transcription factors

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ePlants response to cold stress involves massive regulation of cold responsive genes such as

COR (COR COR78RD29A COR47 COR15a COR66) KIN (cold-induced) LTI (low-

temperature induced) RD (responsive to dehydration) Dehydrin (DHN) Late

EmbryogenesisndashAbundant (LEA) and Responsive To Abscisic Acid (RAB) [3] The

expression of these cold responsive genes is controlled by TFs A set of genes controlled by a

TF is known as a regulon These regulons are regulated either in an ABA-independent or

ABA-dependent manner

311 ABA-independent cold signaling pathway

ABA-independent signalling pathway includes (1) the CBFDREB regulon and (2) the NAC

(NAM ATAF and CUC) and ZF-HD (zinc-finger homeodomain) regulon CBF regulon is

best characterized in cold stress while NAC and ZF-HD regulon is mostly analyzed in

drought and salinity stress [20]

CBFDREB1s bind to C-repeatdehydration-responsive elements (CRTDRE) present in the

promoter of the COR genes and activate transcription CBFs belong to the ethylene

responsive element binding proteinsAPETALA2 family Three cold-responsive CBFs

(CBF1DREB1b CBF2DREB1c and CBF3DREB1a) were identified in Arabidopsis [21]

CBF1 and CBF3 transcripts are positively regulated by NO whereas CBF2 expression

remained unaltered in Arabidopsis [14] In tomato out of the three CBFs (LeCBF1-3) only

LeCBF1 is cold induced Application of SNP increased LeCBF1 expression whereas

nitroarginine (NOS competitive inhibitor) reduced its expression indicating involvement of

NOS-like enzyme in enhancing the expression of LeCBF1 [13] Apart from CBFs Cold

Regulated 15a gene (COR15a) Low temperature induced gene 30 (LTI30) and Low

temperature induced gene 78 (LTI78) are positively regulated by NO [14] suggesting

regulation of both cold induced TFs as well as genes by NO

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eperoxidase (APX) monodehydroascorbate reductase (MDHAR) glutathione reductase (GR)

and dehydroascorbate reductase (DHAR) Cold stress significantly increases the activities of

all the enzymes of ascorbate-glutathione cycle while NO scavenger (2-(4-Carboxyphenyl)-

4455-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and tungstate decreased their activity

in cold treated B ramiflora seeds [16] Besides increasing the enzymatic activities NO also

promote the production of the substrates (ascorbate and GSH) of ascorbate-glutathione cycle

in cold stress S-nitroso-N-acetylpenicillamine (SNAP a NO donor) increased GSH

accumulation while cPTIO and tungstate showed the reversal [16] Similarly Sodium

Nitroprusside (SNP a NO donor) increased ascorbate while cPTIO and L-NAME decreased

its content [11] These results clearly showed the role of NO in positively regulating

ascorbate-glutathione cycle in cold stress Activity of catalase (CAT involved in the

conversion of H2O2 into H2O and O2) is increased by SNP in cold [11] Decreased H2O2

content further confirmed the role of NO in H2O2 removal in cold [11] SNP activated

Superoxide dismutase (SOD remove superoxide radicals) also reaffirmed the role of NO in

ROS removal in cold stress

All these studies convincingly showed that cold induced NO helps in combating cold stress-

induced oxidative damage by increasing the activities of antioxidant enzymes leading to

accumulation of antioxidants which in turn decrease ROS levels (H2O2 and superoxide

content) NO also helps in maintaining the proline level Proline acts as osmolyte stabilizes

the cell membranes and scavange free radicles and thereby contributes to the cold tolerance

Application of tungstate L-NNA and cPTIO led to lesser cold induced proline accumulation

in Arabidopsis leaves [15] and Camelia sinsnesis pollen tubes [17] Also nia1nia2 a NR null

mutant of Arabidopsis was less freezing tolerant reiterating significance of NR generated NO

in cold tolerance due to lesser accumulation of proline Endogenous level of proline is

maintained by P5CS1 (proline synthase) and ProDH (proline dehydrogenase) two key

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eenzymes involved in Pro biosynthesis and degradation respectively Interestingly the

expression of P5CS1 was lower while the expression of ProDH was higher in the mutants

[15] In addition Viagra (an inhibitor of cyclic guanosine monophosphate (cGMP) specific

phosphodiesterase used to stimulate cGMP level) promoted proline accumulation and the

effect was partially reversed with L-NNA or cPTIO in cold [17] Therefore suggesting the

role of cGMP as a downstream target of NO mediated proline accumulation in cold stress

NO also contributes to S-nitrosothiols (SNOs) and GSNO formation which are the

endogenous reservoir of NO [18] Cold stress enhanced SNO accumulation up to 12 fold (4

degC 6 h) in B juncea seedlings [7] 5 fold (48 h 8 ordmC) in P sativum leaves [8] and 2 fold in B

ramiflora embryos [16] Confocal laser scanning microscopy (CLSM) further confirmed

SNO accumulation in vascular tissue palisade and spongy mesophyll after cold stress (1 d 8

degC) in C annum leaves [9] The effect of cold stress on GSNO content is not investigated yet

but increased activity of GSNO reductase (GSNOR) was observed in pepper leaves in cold

[9] indicating GSNO accumulation GSNOR catalyzes the reduction of GSNO to glutathione

disulfide (GSSH) and ammonia (NH3) GSNOR provides cellular protection against

nitrosative stress in cold stress by maintaining low endogenous levels of GSNO [19]

It is well established that cold stress induced NO production either by oxidative or reductive

pathways contributes to cellular storage pool in the form of GSNO and SNO which in turn act

as a depot of NO for its sustained release and reaction with varied types of enzymes including

antioxidant enzymes Besides it also modulates genes thus playing an important role in

modulating the cold stress responsive transcriptome

3 Transcriptional and posttranslational modulation of cold responsive transcriptome

and Transcription factors

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ePlants response to cold stress involves massive regulation of cold responsive genes such as

COR (COR COR78RD29A COR47 COR15a COR66) KIN (cold-induced) LTI (low-

temperature induced) RD (responsive to dehydration) Dehydrin (DHN) Late

EmbryogenesisndashAbundant (LEA) and Responsive To Abscisic Acid (RAB) [3] The

expression of these cold responsive genes is controlled by TFs A set of genes controlled by a

TF is known as a regulon These regulons are regulated either in an ABA-independent or

ABA-dependent manner

311 ABA-independent cold signaling pathway

ABA-independent signalling pathway includes (1) the CBFDREB regulon and (2) the NAC

(NAM ATAF and CUC) and ZF-HD (zinc-finger homeodomain) regulon CBF regulon is

best characterized in cold stress while NAC and ZF-HD regulon is mostly analyzed in

drought and salinity stress [20]

CBFDREB1s bind to C-repeatdehydration-responsive elements (CRTDRE) present in the

promoter of the COR genes and activate transcription CBFs belong to the ethylene

responsive element binding proteinsAPETALA2 family Three cold-responsive CBFs

(CBF1DREB1b CBF2DREB1c and CBF3DREB1a) were identified in Arabidopsis [21]

CBF1 and CBF3 transcripts are positively regulated by NO whereas CBF2 expression

remained unaltered in Arabidopsis [14] In tomato out of the three CBFs (LeCBF1-3) only

LeCBF1 is cold induced Application of SNP increased LeCBF1 expression whereas

nitroarginine (NOS competitive inhibitor) reduced its expression indicating involvement of

NOS-like enzyme in enhancing the expression of LeCBF1 [13] Apart from CBFs Cold

Regulated 15a gene (COR15a) Low temperature induced gene 30 (LTI30) and Low

temperature induced gene 78 (LTI78) are positively regulated by NO [14] suggesting

regulation of both cold induced TFs as well as genes by NO

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eenzymes involved in Pro biosynthesis and degradation respectively Interestingly the

expression of P5CS1 was lower while the expression of ProDH was higher in the mutants

[15] In addition Viagra (an inhibitor of cyclic guanosine monophosphate (cGMP) specific

phosphodiesterase used to stimulate cGMP level) promoted proline accumulation and the

effect was partially reversed with L-NNA or cPTIO in cold [17] Therefore suggesting the

role of cGMP as a downstream target of NO mediated proline accumulation in cold stress

NO also contributes to S-nitrosothiols (SNOs) and GSNO formation which are the

endogenous reservoir of NO [18] Cold stress enhanced SNO accumulation up to 12 fold (4

degC 6 h) in B juncea seedlings [7] 5 fold (48 h 8 ordmC) in P sativum leaves [8] and 2 fold in B

ramiflora embryos [16] Confocal laser scanning microscopy (CLSM) further confirmed

SNO accumulation in vascular tissue palisade and spongy mesophyll after cold stress (1 d 8

degC) in C annum leaves [9] The effect of cold stress on GSNO content is not investigated yet

but increased activity of GSNO reductase (GSNOR) was observed in pepper leaves in cold

[9] indicating GSNO accumulation GSNOR catalyzes the reduction of GSNO to glutathione

disulfide (GSSH) and ammonia (NH3) GSNOR provides cellular protection against

nitrosative stress in cold stress by maintaining low endogenous levels of GSNO [19]

It is well established that cold stress induced NO production either by oxidative or reductive

pathways contributes to cellular storage pool in the form of GSNO and SNO which in turn act

as a depot of NO for its sustained release and reaction with varied types of enzymes including

antioxidant enzymes Besides it also modulates genes thus playing an important role in

modulating the cold stress responsive transcriptome

3 Transcriptional and posttranslational modulation of cold responsive transcriptome

and Transcription factors

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ePlants response to cold stress involves massive regulation of cold responsive genes such as

COR (COR COR78RD29A COR47 COR15a COR66) KIN (cold-induced) LTI (low-

temperature induced) RD (responsive to dehydration) Dehydrin (DHN) Late

EmbryogenesisndashAbundant (LEA) and Responsive To Abscisic Acid (RAB) [3] The

expression of these cold responsive genes is controlled by TFs A set of genes controlled by a

TF is known as a regulon These regulons are regulated either in an ABA-independent or

ABA-dependent manner

311 ABA-independent cold signaling pathway

ABA-independent signalling pathway includes (1) the CBFDREB regulon and (2) the NAC

(NAM ATAF and CUC) and ZF-HD (zinc-finger homeodomain) regulon CBF regulon is

best characterized in cold stress while NAC and ZF-HD regulon is mostly analyzed in

drought and salinity stress [20]

CBFDREB1s bind to C-repeatdehydration-responsive elements (CRTDRE) present in the

promoter of the COR genes and activate transcription CBFs belong to the ethylene

responsive element binding proteinsAPETALA2 family Three cold-responsive CBFs

(CBF1DREB1b CBF2DREB1c and CBF3DREB1a) were identified in Arabidopsis [21]

CBF1 and CBF3 transcripts are positively regulated by NO whereas CBF2 expression

remained unaltered in Arabidopsis [14] In tomato out of the three CBFs (LeCBF1-3) only

LeCBF1 is cold induced Application of SNP increased LeCBF1 expression whereas

nitroarginine (NOS competitive inhibitor) reduced its expression indicating involvement of

NOS-like enzyme in enhancing the expression of LeCBF1 [13] Apart from CBFs Cold

Regulated 15a gene (COR15a) Low temperature induced gene 30 (LTI30) and Low

temperature induced gene 78 (LTI78) are positively regulated by NO [14] suggesting

regulation of both cold induced TFs as well as genes by NO

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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ePlants response to cold stress involves massive regulation of cold responsive genes such as

COR (COR COR78RD29A COR47 COR15a COR66) KIN (cold-induced) LTI (low-

temperature induced) RD (responsive to dehydration) Dehydrin (DHN) Late

EmbryogenesisndashAbundant (LEA) and Responsive To Abscisic Acid (RAB) [3] The

expression of these cold responsive genes is controlled by TFs A set of genes controlled by a

TF is known as a regulon These regulons are regulated either in an ABA-independent or

ABA-dependent manner

311 ABA-independent cold signaling pathway

ABA-independent signalling pathway includes (1) the CBFDREB regulon and (2) the NAC

(NAM ATAF and CUC) and ZF-HD (zinc-finger homeodomain) regulon CBF regulon is

best characterized in cold stress while NAC and ZF-HD regulon is mostly analyzed in

drought and salinity stress [20]

CBFDREB1s bind to C-repeatdehydration-responsive elements (CRTDRE) present in the

promoter of the COR genes and activate transcription CBFs belong to the ethylene

responsive element binding proteinsAPETALA2 family Three cold-responsive CBFs

(CBF1DREB1b CBF2DREB1c and CBF3DREB1a) were identified in Arabidopsis [21]

CBF1 and CBF3 transcripts are positively regulated by NO whereas CBF2 expression

remained unaltered in Arabidopsis [14] In tomato out of the three CBFs (LeCBF1-3) only

LeCBF1 is cold induced Application of SNP increased LeCBF1 expression whereas

nitroarginine (NOS competitive inhibitor) reduced its expression indicating involvement of

NOS-like enzyme in enhancing the expression of LeCBF1 [13] Apart from CBFs Cold

Regulated 15a gene (COR15a) Low temperature induced gene 30 (LTI30) and Low

temperature induced gene 78 (LTI78) are positively regulated by NO [14] suggesting

regulation of both cold induced TFs as well as genes by NO

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eInducer of CBF expression 1 (ICE1) is an upstream activator of CBF3 [22] High expression

of osmotically responsive genes1 (HOS1 a RING fingerndashcontaining E3 ubiquitin ligase) and

AtSIZ1 (SUMO E3 ligase) are involved in the ubiquitinization [23] and sumolyation [21] of

ICE1 respectively Recently SIZ1 was identified as an in vivo tyrosine nitrated target in

Arabidopsis [24] but it is yet to be functionally validated ZAT12 a C2H2 zinc finger protein

is a negative regulator of CBFs [25] However its expression was not affected by NO [14]

Plant nonsymbiotic haemoglobin (Hb1) also regulates CBF pathway [14] Plant haemoglobin

is either symbiotic (leghemoglobin) or non-symbiotic Non-symbiotic haemoglobin has two

classes class 1 (Hb1) and class 2 (Hb2) on the basis of differential oxygen binding affinities

Hb1 metabolize NO to nitrate using NADPH as an electron donor [26] Arabidopsis Hb over-

expressing lines (AHb1) showed reduced NO due to its scavenging by Hb1 and also showed

decreased expression of CBF1 and CBF3 in Arabidopsis [14] Earlier transgenic lines of

Lycoperscicon esculentum A thaliana Brassica napus Nicotiana tabacum and Triticum

aestivum with improved cold tolerance were generated by over expressing CBF and COR15

gene [25] The strategy of silencing Hb1 to enhance NO production and to positively regulate

CBF regulon could be tried as an alternate strategy to engineer crops with improved cold

tolerance

312 ABA-dependent cold signaling pathway

ABA-dependent signalling pathway consists of two regulons namely (1) the AREBABF

regulon and (2) the MYCMYB regulon [20] ABA induces the expression of cold regulated

genes by promoting the binding of bZIP TFs to AREBs The effect of NO on the DNA-

binding activity of AtMYB2 (a R2R3-MYB TF from A thaliana which regulate MYCMYB

regulon) was analyzed [27] A fully active minimal DNA-binding domain of AtMYB2

spanning residues 19ndash125 referred as M2D was cloned Using electrophoretic mobile gel

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eshift assay (EMSA) the binding of M2D to the core binding site 50-[A]AACC[A]-30 MYB

was shown SNP and GSNO inhibited M2D DNA-binding and the effect was reversed by

DTT (a thiol specific reductant) S-nitrosylation of M2D (at Cys53) was shown by biotin

switch technique These results showed that the DNA-binding of M2D is inhibited by S-

nitrosylation suggesting negative regulation of the MYB dependent signalling by NO

To summarize ABA-independent cold stress signalling pathway is regulated by modulation

of CBF1 CBF3 COR15a LTI30 and LTI78 by NO Alternatively CBFs can also be

modulated by manipulating Hb1 expression The ABA-dependent cold stress signalling

pathway is negatively regulated by S-nitrosylation of AtMYB2

Besides modulating the cold responsive genes and TFs as described NO also modifies

proteins by PTMs thus it would be important to understand the extent to which NO modulates

cold responsive proteome For analysing the effect of cold stress either the proteome of

control tissue is compared with that of cold treated tissue or the proteome of a cold tolerant

variety is compared with a cold sensitive one As the focus of this review is to give an

overview on the NO-cold stress signaling the techniques which are utilized to identify cold

stress and NO based PTMs modulated proteins are briefly described For details the reviews

on the methods for the identification of NO based PTMs (S-nitrosylation [28 29] S-

gluthathionylation [30] tyrosine nitration [31]) can be referred

4 Approaches to identify cold responsive and NO modulated targets

For the detection of the S-nitrosylated proteins Biotin Switch Technique (BST) and its

modified variants are used (Figure 2) [32] It involves three basic steps blocking of free

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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ethiols in the proteins by methyl methanethiosulfonate (MMTS) or N-ethylmaleimide (NEM)

followed by the reduction of S-nitrosylated cysteine residues with ascorbate After that the

reduced cysteine residues are reacted with biotin-HPDP (N-[6-(biotinamido) hexyl]-3prime-(2prime-

pyridyldithio)-propionamide) a process known as biotinylation Biotinylation helps in the

detection of S-nitrosylated proteins either by western blotting using anti-biotin antibodies or

in the purification by neutravidin affinity chromatography Purified S-nitrosylated proteins

are identified by LCndashMSMS In SNOSID (SNO site identification) the biotinylated proteins

are digested before affinity purification [33] His-Tag Switch is a modification of BST where

cysteine residues are attached with His-containing peptide instead of biotin [34] Fluorescent

dyes such as DyLight maleimide sulfhydryl reactive fluorescent compounds cyanine

maleimide sulfhydryl reactive compounds and 7-amino-4-methyl coumarin-3-acetic-acid

(AMCA)-HPDP are also used for the relative quantification of the S-nitrosylated proteins on

2-D gels in control and stressed samples [35 36] SNO-RAC (S-nitrosothiols using resin-

assisted capture) helps in site-specific identification of the modified cysteine residues by LCndash

MSMS [37] d-Switch is another modification of BST which allows the quantitative

assessment of the S-nitrosylation [38] Gold nanoparticle (AuNP) based technique is useful

for the identification of the both S-nitrosylation and S-glutathionylaton site (also known as S-

modifications) but due to the affinity of AuNPs particles to both S-nitrosylated and S-

glutathionylated sites it makes the identification less specific [39] In phosphine-based

method blocking step of the traditional BST is omitted which helps in reducing the false

positive results due to incomplete blocking of the free thiols [40] Organomercury-based

capture involves a reaction between phenylmercury compounds (either conjugated to an

agarose solid support or to polyethylene glycol-biotin) with S-nitrosocysteine residues to

form a stable thiol-mercury bond S-nitrosylation site could be identified using this approach

[41]

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eS-glutathionylated proteins are detected by western blotting immunocytolocalization and

immunoprecipitation using anti-glutathione antibodies in vivo S-glutathionylated proteins are

identified by glutathione radiolabeling and visualized by autoradiography or phosphor

imaging technologies [42] Biotinylated glutathione has advantage over radiolabeling as low

abundant targets could be identified [43] GRX reduction [44] and glutathione-S-transferase

(GST) overlay [45] methods are also used to purify and identify S-glutathionylated proteins

Tyrosine nitrated proteins are predominantly detected and in situ localized using 3-

nitrotyrosine (NO2-Tyr) antibodies [46] Purified nitrated proteins are resolved on 2-DE and

identified by MS [24] Gas chromatographynegative chemical ionization tandem mass

spectrometry [47] and Combined fractional diagonal chromatography (COFRADIC) [48] are

recently developed techniques which allow the quantitative assessment and identification of

the nitrated sites These two techniques are not yet utilized in plants

For analysing the cold modulated proteome and NO based PTMs 2-DGE MS has been

routinely used MS is also used to quantitate the differential abundance of proteins either by

protein labelling (iTRAQ) or by label free methods A comparative analysis showed that the

label-free approach resulted in identification of 236 cold modulated targets while iTRAQ

could identify only 85 cold responsive proteins in rice [49] As gel free approach is amenable

to automation it is a preferred approach to minimise the variations arising due to manual

handling After identification the targets are validated for cold and NO specific modulation

either by activity assaystaining or western blotting Majority of the techniques described in

the above sections are recently developed which indicates that this area of research has

gained a lot of interest in last 4-5 years Although the techniques developed in the past has

contributed a lot to the understanding of NO signaling additional work is needed to improve

the sensitivity and the specificity of these techniques In future the focus should be on the

identification of the ldquosite of NO modificationrdquo in the proteins Also it would be important to

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eidentify the factors which govern the selectivity of the modification For examples S-

nitrosylated tyrosine nitrated and carbonylated proteins identified in salt stress in citrus

showed some proteins (10-formyltetrahydrofolate synthetase sulfite reductase and thiazole

synthase) to be specifically modified by only one modification whereas some of these

(RuBisCO large subunit glutamine synthetase and actin) were modified by all the three

modifications [50] The PTMs cross-talk also needs to be dissected and understood in

physiological context In addition NO based PTMs analysis in sub-cellular system could

identify novel regulatory mechanism

To extend this NO-cold stress cross-talk further all reported cold responsive proteins were

analysed for their modification by NO based PTMs

5 Proteomic evidences for cold and NO signalling cross talk

Proteome of an organism in not uniquely directed by its genome due to the existence of

alternative splicing protein-protein interactions and PTMs Therefore analysis of the

proteins the real executors and final reflectors of the gene expression can provide better

picture of the signalling mechanisms operating in stress Although there are good number of

reports on analysis of cold responsive proteomes information is scanty about their PTMs in

cold stress PTMs can either inhibit or activate the enzymes modulate DNA-binding of the

TFs or re-localize the proteins Till date there are only three studies detailing NO based PTMs

of proteins in cold Therefore to have a broader view on NO modulation of cold responsive

proteins complete repertoire of these was analysed for their S-nitrosylation tyrosine nitration

andor S-glutathionylation to understand the potential association between these PTMs and

cold stress signaling

51 Cold stress promotes NO based Post Translational modifications

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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eS-nitrosoproteome analysis of B juncea in cold stress showed cold induced differential S-

nitrosylation of defence related (cysteine-rich repeat secretory protein and major latex like

proteins) signaling (PpX-GppA phosphatase) metabolic (fructose bisphosphate aldolase) and

photosynthetic proteins (ribulose-15-bisphosphate carboxylaseoxygenase RuBisCO) [9] S-

nitrosylation inactivated RuBisCO which correlated with decreased photosynthetic

efficiency Repeated identification of RuBisCO as S-nitrosylated S-glutathionylated and

nitrated target suggest significant role of NO in modulation of RuBisCO activity the main

CO2 fixing enzyme in photosynthesis Similar extent of inhibition by GSNO and cold stress

of RuBisCO activity suggested that cold modulated S-nitrosylation is responsible for

RuBisCO inhibition which in turn could explain the cold stress-induced photosynthetic

inhibition [7]

Cold stress induced protein tyrosine nitration in P sativum showed six immunoreactive

nitrated polypeptides (29-59 kDa) [8] Three polypeptides (59 kDa 42 kDa and 29 kDa)

showed higher nitration in comparison with the control while nitration of a 73 kDa

polypeptide was specifically induced by cold Similarly in C annum leaves increased

nitration of four polypeptides (45-97 kDa) was observed after cold stress (1 d) which

decreased to negligible levels after 3rd

day [9] Unfortunately none of the cold responsive

nitrated protein is identified and validated till date Furthermore S-glutathionylation analysis

in cold stress is yet to be analyzed

52 Cold responsive proteins related to sugar metabolism ROS detoxification and

defense are significantly modulated by NO

Effect of cold stress was analysed in Oryza sativa T aestivum Festuca pratensis A

thaliana P sativum Lathyrus sativus Helianthus annuus Thellungiella halophila Nicotiana

tabacum Solanum tuberosum Poplar Picea obovata Physcomitrella patens Chicory Zea

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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emays S lycopersicum Glycine max Prunus persica Strawberry and Cotton as summarized

in Table 1 To understand the differential spatial responses to cold stress the proteomes of

leaves roots anther seed bark cotton fibre and fruits were also analysed Leaves are the

major site for CO2 fixation and assimilation As leaf proteins are majorly involved in the

photosynthesis and sugar metabolism any change in the leaf proteome therefore would

affect the crop yield Leaf proteomes of different plants showed 400-1700 spots in silver

stained or CBB stained 2-D gels Overall significant proportion 35-21 of the leaf

proteome responded to cold stress suggesting its vast reprogramming The cold responsive

proteins were majorly involved in protein stabilization protein degradation signalling

energy metabolism antioxidative pathways cell wall modifications and defence responses

Analysis of cold responsive root proteome in rice [51 52] and chicory [53] showed

metabolism ROS detoxification and defense functions to be affected suggesting contribution

of these to cold response and adaptation

Identification of cold modulated peach bark indicated reprogramming of energy metabolism

carbohydrate metabolism defence electron transport and cytoskeleton organization Analysis

of cold responsive anther proteins from maize [54] and rice [55 56] showed their

participation in ROS detoxification and carbohydrate metabolism Soybean seed proteome

analysis showed increased abundance of storage and defence related proteins in cold [57]

Similarly cotton fibre proteome showed specific modulation of sugar metabolism cell wall

loosening cellulose synthesis cytoskeleton and redox regulation as a cold stress response

[58]

A comparative analysis of the cold responsive proteins from different tissues (leaf root

anther and fruit Figure 2A) clearly showed that metabolic proteins are most affected by cold

stress indicating extensive reprogramming of the metabolome in cold stress After

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

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e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

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emetabolism ROS related enzymes were the second most affected category suggesting ROS

detoxification to be another important component in providing cold tolerance In addition

accumulation of defencestress related targets was common in all the studies indicating their

pivotal role in cold stress tolerance Unfortunately targets related to signalling were least

identified suggesting a need for the deep proteome analysis for enrichment of these targets

and better understanding of the cold stress signalling networks

NO based modifications of all the cold responsive proteins from redox defensestress and

signaling categories showed 59 of the redox related targets to be either S-nitrosylated

nitrated andor S-glutathionylated (Figure 2B) This predicts a significant role of NO in

regulating the redox status in stress Similarly 50 of defence related targets were modified

by NO based PTMs reflecting the role of NO signalling in defence responses Around 30 of

the cold responsive signaling related proteins were modified by NO posttranslationally To

have an overview of the regulatory role(s) of NO signaling in cold stress comprehensive

knowledge of these PTMs for all the cold responsive proteins is essential These potential NO

modulated targets need to be verified at the biochemical molecular and physiological levels

In addition it would be very important to know whether these proteins are modulated by NO

in vivo

In order to understand the physiological impact of this NO-cold stress cross-talk all reported

cold responsive proteins were analysed for NO based PTMs It was important to

analysepredict their potential impact on major metabolic pathways which are significantly

perturbed in cold stress to provide cold tolerance

521 NO based PTMs modulate majority of sugar metabolising enzymes

Cold stress results in a readjustment of sugar metabolism resulting in their accumulation

during cold acclimation Analysis of information on cold responsive proteins clearly

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

Acc

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

epte

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

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d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

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d A

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eindicated major pathways of sugar metabolism including glycolysis Krebrsquos cycle and pentose

phosphate pathway to be mostly up-regulated by cold stress (Figure 3) Except for Glucose-6-

phosphate isomerase and pyruvate kinase all other enzymes of glycolysis mainly showed

increased abundance by cold Interestingly cytosolic glyceraldehyde-3-phosphate

dehydrogenase which converts glyceraldehydes 3-phosphate to D-glycerate 13-biphosphate

is tightly regulated by NO as it is S-nitrosylated and nitrated in salinity stress in Citrus

aurantium leaves [50] However in pentose phosphate pathway glucose-6-phosphate

dehydrogenase transketolase and gluconate dehydrogenase are the only enzymes which

respond to cold stress (Figure 3) Cold responsive Krebrsquos cycle enzymes include citrate

synthase aconitate hydratase isocitrate dehydrogenase and malate dehydrogenase Several

other enzymes including sucrose synthase 1 and 2 phosphoglucomutase UDP-glucose

pyrophosphorylase and UDP-glucose-4-epimerase (UGE) are also modulated by cold stress

(Figure 3) Sucrose synthase 1 and UDP-glucose pyrophosphorylase produce UDP-glucose a

precursor for cellulose synthesis which leads to cell wall hardening during cold stress

However modulation of these targets by NO is not known

These alterations in sugar metabolizing enzymes result in the accumulation of sucrose

raffinose trehalose maltose glucose and fructose contributing to maintaining the osmoticum

of the cell decreasing the ice nucleation temperature [59] and some like fructose-6-phosphate

and fructose bisphosphate exhibit ROS scavenging property thus preventing the oxidative

damage [60]

On the basis of these studies it is concluded that 88 80 and 50 of cold modulated

targets of glycolysis Krebrsquos cycle and pentose phosphate pathways are modulated by NO

post-translationally confirming the significance of these PTMs in regulating sugar

metabolism

Acc

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e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

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e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

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edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

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eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

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eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

e522 The detoxification machinery and redox status are also modified by NO based

PTMs

It is well established that enzymatic activities of antioxidant enzymes are increased by NO

(section 2) Interestingly these enzymes are also post-translationally modified CAT [11] and

SOD [11 61] showed increased abundance as well as activities in cold stress These enzymes

are S-nitrosylated and nitrated after salt stress and pathogen infection (Table 2) Other ROS

related cold modulated enzymes which aid in ROS detoxification includes 2-

cysperoxiredoxinperoxiredoxin [61 62 63] thioredoxin [61 64] and ferretin Cysteine

peroxiredoxin and thioredoxin are S-nitrosylated after pathogen infection while MDHAR is

S-nitrosylated in salt stress (Table 2) Interestingly DHAR is S-nitrosylated [65] S-

glutathionylated [43] and nitrated [50] suggesting a possible cross-talk of all the three NO

based PTMs in its regulation

523 Cold stress modulated defencestress associated proteins are also regulated by NO

Plants adapt to cold stress by accumulating the stress related proteins as cold stress makes

them prone to the infection by the psychrophyllic pathogens Nine categories of PR

(pathogenesis-related) proteins including PR2 (β-13-glucanase) [66 67 68] PR3 (chitinase)

[61 68 69] PR4 [68] PR5 (thaumatin like protein) [61 68 70 71] PR10a [72] PR11 (class

I chitinase) [73] PR14 (lipid transfer protein) [74] PR17 [72] and intracellular pathogenesis-

related protein like are cold responsive Recently S-nitrosylation of PR 10 was reported in

the S tuberosum tuber [75] Chitinases are involved in the degradation of cell wall of fungus

however chitinase of class I and class II acquire antifreeze activities during cold stress

Chitinase have been repeatedly identified as cold responsive target Interestingly Chitinases

were also reported to be in vivo S-nitrosylated and nitrated in salt stress [50 75 76 77]

Acc

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eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

Acc

epte

d A

rticl

e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

Acc

epte

d A

rticl

edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

Acc

epte

d A

rticl

eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

Acc

epte

d A

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e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

Acc

epte

d A

rticl

eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

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eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

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e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

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eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

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eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

eIn addition to these PR-proteins different isoforms of dehydrins [53 68 73 78] including

COR15 [73] cold acclimation protein WCOR615 [78] dehydrin ERD10 [66] COR18 [73]

also accumulate in cold Dehydrins being hydrophilic cryoprotect the membranes and

enzymes [79] Dehydrins of Prunus persica and Forsythia suspensa possess antifreeze

activity [80 81] Besides Hara et al showed ROS scavenging activity of CuCOR19 a

dehydrin [82] COR15a and COR18 are S-nitrosylated in Arabidopsis [Table 2 83] NO also

enhanced transcription of COR15a gene [14] thus indicating its dual regulation at both the

transcriptional and post-translational level

Glyoxalase 1 showed increased abundance during cold stress [51 61 68 73] Glyoxalase 1

detoxifies methylglyoxal which is formed as a by-product of carbohydrate and amino acid

metabolism Recently nitration [50] and S-nitrosylation (unpublished data) of Gly 1 was

shown in salinity and cold stress respectively

524 Modification of signalling related proteins calmodulin peptidyl prolyl cis-trans

GTP-binding nuclear protein Ran-1 and 14-3-3 proteins by NO

Cold modulated signaling components identified in 2-DE-MS based studies includes

RAB21A [57] RAB2A [63] Rab-type small GTP-binding protein Ethylene-responsive

GTP-binding protein G-protein beta-subunit-like protein [67] Putative RAB24 Protein [84]

and Ras-related small GTP-binding protein RAB1c [85] GTP-binding nuclear protein Ran-1

is nitrated in citrus [50]

Cold responsive components of calcium signalling include 14-3-3 protein [72] Calcium-

binding protein [49] and probable calmodulin-like protein-7 [49] S-nitrosylation [77] and

nitration [86] of calmodulin suggested interaction of NO with this calcium dependent

signalling component 14-3-3 protein was identified as a heat responsive nitrated target in

sunflower [46] Peptidyl prolyl cis-trans isomerase was S-nitrosylated in cold stress

Acc

epte

d A

rticl

e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

Acc

epte

d A

rticl

edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

Acc

epte

d A

rticl

eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

Acc

epte

d A

rticl

e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

Acc

epte

d A

rticl

eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

epte

d A

rticl

eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

e(unpublished) in B juncea seedlings and tyrosine nitrated in salt stress in citrus (Table 2)

[50]

Above mentioned analysis of major metabolic pathways which are reprogrammed to provide

cold stress tolerance suggest a strong involvement of NO based PTMs as a regulatory

mechanism Although these studies support strong cross-talk between NO and cold stress

signalling pathways but to understand the spatial dimension of the cold stress signalling it is

pertinent to analyse the cold stress signalling for organelles

6 Spatial nitric oxide and cold stress cross talk at sub-cellular level

Due to ease in protein extraction procedures and advancement in MS technologies now it is

possible to analyse an organelle proteome to have spatial distribution of the proteins and

regulating mechanisms Almost all the major cold responsive organelles proteomes including

mitochondria chloroplast nucleus along with apoplast and plasma membrane have been

analysed NO signalling in subcellular proteome is described as per their spatial arrangement

in the cell ie right from the apoplast to plasma membrane followed by mitochondria and

chloroplast and finally in the nucleus

61 Cold stress induces accumulation of antifreeze and defence related proteins some

of which are S-nitrosylated in the apoplast

Apoplast is the region between the cell wall and plasma membrane Effect of cold stress on

apoplast proteomesecretome was analyzed in H rhamnoides [69] Apoplastic proteins from

cold treated seedlings resolved as 245 spots in silver stained 2-D gels Identification of

proteins showed their association with defence redox regulation and signalling Antioxidant

enzymes like SOD showed a decrease and peroxidase showed enhanced activity with NO

Acc

epte

d A

rticl

edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

Acc

epte

d A

rticl

eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

Acc

epte

d A

rticl

e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

Acc

epte

d A

rticl

eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

epte

d A

rticl

eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

edonor in wheat apoplast [86] suggesting its role in maintaining the redox status in the

apoplast In B juncea apoplast myrosinase DHAR cysteine protease and germin-like

proteins were S-nitrosylated in cold stress (unpublished data) Cold stress also results in the

secretion of antifreeze proteins in the apoplast Antifreeze proteins bind to the ice crystals and

retard their growth However regulation of the antifreeze protein by NO is not yet known

therefore it would be an important area of research in future Also for the better

understanding of NO-cold stress cross-talk identification of other NO based PTMs in

apoplast would be important

62 Regulation of membrane and cytoskeletal proteins by cold and NO

After the cell wall plasma membrane is in the next contact with the environment It helps in

sensing the temperature Cold modulated plasma membrane proteomes were analysed in

Arabidopsis [61] and rice [70] Identified proteins were mainly associated with membrane

repair membrane protection proteolysis CO2 fixation energy production signal

transduction protein synthesis cell division and defence Interestingly Glycine-rich RNA-

binding protein a cold modulated target was identified as an in vivo nitrated in Arabidopsis

seedlings [24] and S-nitrosylated target [75 83] in potato leaves and Arabidopsis leaves

Similarly S-nitrosylation of annexin another cold modulated plasma membrane protein is

shown in rice [83]

NO controls numerous cystoskeleton mediated processes such as root growth vesicle

trafficking and guard cell movements [87] Cystosketal proteins like α-tubulin and actin are

tyrosine nitrated [24] S-nitrosylated [83] and S-glutathionylated [43]

Tyrosinationdetyrosination cycle and S-nitrosylation helps in maintaining the dynamics of

microtubules thus allowing the cells to respond to intracellular and extracellular signals It is

Acc

epte

d A

rticl

eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

Acc

epte

d A

rticl

e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

Acc

epte

d A

rticl

eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

epte

d A

rticl

eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

eclear that NO based PTMs affectmodulate the cytoskeletal proteins and thus might be

involved in the cold signal perception

63 The chloroplast lumen proteome is less responsive to cold stress than stromal

proteome

Chloroplast is the site of photosynthesis an important assimilatory process which is

responsible for the food production Cold modulated plastid proteome was analysed in

Arabidopsis after cold stress treatments (5 degC for 1 10 and 40 days) [62] 2D-DIGE gels

showed major changes in both stromal and luminal proteomes after 40 days of cold stress

Identified proteins were mainly associated with photosynthesis and redox regulation

A close examination of the identified cold responsive chloroplastic proteins as detailed in

Supplementary Table 2 showed the regulation of the enzymes of both the light and dark

reactions of photosynthesis by NO based PTMs As the photosynthetic machinery of the

chloroplast is a source of ROS the chloroplastic proteins are direct targets of oxidative

modifications in cold S-glutathionylated thioredoxin was proposed to down-regulate Calvin-

Benson cycle in oxidative stress which indicated indirect regulation of photosynthesis by NO

[88] S-glutathionylation of the choroplastic GAPDH appeared as a protective mechanism

against oxidative stress Chloroplastic thioredoxin (f and m type) were identified to be S-

nitrosylated [76] and S-glutathionylated [89]

64 In mitochondrial proteome cold stress degrades key matrix enzymes increases

abundances of heat shock proteins and S-nitrosylation inhibited the activity of P

proteins of GDC complex

Mitochondrial proteome was analysed in pea after cold stress (4 degC 36 h) in combination

with drought and herbicide treatment [90] Mitochondrial protein resolved on 2-DGE showed

Acc

epte

d A

rticl

e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

Acc

epte

d A

rticl

eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

epte

d A

rticl

eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

e33 cold responsive spots Except for both the subunits of RuBisCO β-carbonic anhydrase and

CuZn-SOD which were chloroplastic and cytoplasmic contaminations rest 29 proteins were

mitochondrial Degradation of H P T and L subunits of glycine decarboxylase (GDC) was

observed after stress Chilling degraded more key matrix enzymes in comparison with other

two stress conditions (drought and herbicide stress) Abundance of F1F0 ATP synthase

subunits increased after stress but these were breakdown products rather than the intact

proteins S-nitrosylation analysis of the GSNO treated mitochondrial fractions from

Arabidopsis leaves identified 11 S-nitrosylated proteins [91] Three subunits (P1 P2 and H1)

of the GDC the key enzyme of C2 cycle were S-nitrosylatedS-glutathionylated Validation

of purified P protein for S-nitrosylation showed inhibition in the GDC activity with SNP and

a reversal with cPTIO suggesting the inhibition of photorespiratory pathway by S-

nitrosylationS-glutathionylation Mitochondrial NAD-dependent malate dehydrogenase

(MDH) involved in malatendashaspartate shuttle and ATP generation was S-nitrosylated during

the progression of hypersensitive response in Arabidopsis [92] Other mitochondrial S-

nitrosylated targets included succinyl-CoA synthetase and ATP synthase alpha chain [76]

Cold stress also increased abundance of heat shock proteins (HSP90 HSP70 and HSP22)

HSP70 is known to be tyrosine nitrated S-nitrosylated and S-glutathionylated [24 43 83]

It is evident that NO inhibits the photorespiratory pathway in mitochondria Moreover cold

modulated protein degradation in matrix probably leads to accumulation of HSPs

Surprisingly some of these HSPs like HSP70 are heavily modulated by NO mediated PTMs

but the significance of these modifications in mitochondria in cold signalling is presently not

clear

65 Nitrosylation regulates subcellular targeting and oligomerization in nuclear

proteome

Acc

epte

d A

rticl

eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

epte

d A

rticl

eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

eNucleus is the hub of all the regulatory information of the cell Nuclear proteome analysis can

provide insight into altered gene expression after stress [93] Nuclear proteome of

Arabidopsis showed 210 spots out of which 54 were cold responsive Interestingly

transcription factors of ABA-independent and ABA-dependent cold stress signalling

pathways including MYB bZIP bHLH MYB34 and bHLH transcription factor similar to

MYC were identified supporting their roles in cold stress signalling

However few nuclear proteins like AtMYB2 [27] Non-expressor of Pathogenesis Related-1

(NPR1) [94] b-ZIP transcription factor TGA1 [95] are modulated by NO Cold responsive

S-nitrosylated nuclear proteins includes probable WRKY transcription factor [76] D of zinc

finger proteins and GAPDH [96] Binding of S-nitrosylated GAPDH (SNO-GAPDH) to

Siah1 (an E3 ubiquitin ligase) facilitates its translocation from cytoplasm to the nucleus

where Siah1 promotes ubiquitin mediated degradation of the nuclear proteins [97] In

addition SNO-GAPDH mediated transnitrosylation of the nuclear proteins including

deacetylating enzyme (SIRT1) histone deacetylase-2 (HDAC2) and DNA-activated protein

kinase (DNA-PK) of HEK293 and 293T cells suggests participation of NO in the nuclear

signaling [98] Recently interaction of GAPDH with osmotic stress-activated protein kinase

(NtOSAK) along with an increase in its S-nitrosylation in Nicotiana tabacum cell

suspensions in salt stress was shown which suggested its alternate role in plant stress

signaling cascades besides its role in glycolysis [98]

NPR1 is a master regulator of genes involved in the defense responses and plays an important

role in providing plant resistance to stress [99] GSNO facilitates the oligomerization of

Arabidopsis NPR1 through S-nitrosylation of Cys 156 and blocks its nuclear translocation

[94] In addition TGA1 a bZIP transcription factor is S-nitrosylated and S-glutathionylated

[95] Hence NO affects NPR1TGA1 system thus regulating plant defense responses

Acc

epte

d A

rticl

eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

eThese examples leave little doubt that NO mediated PTMs regulate sub-cellular transport

thus could participate in retrograde signalling during stress conditions

Reports on modification of lipids by NO are few but are indicative of NO-cold stress cross-

talk

7 Modulation of cold responsive lipids by NO ndash continues NO-cold stress

cross-talk

It is evident that the interaction of cold stress and NO vis a vis lipid is a novel area of

investigation and therefore the information for lipid modifications by NO in cold stress is

limited Recently NO-lipid signalling interplay was observed when an increase in NO

phosphatidic acid (PtdOH) and two phosphosphingolipid species the long chain base (LCB)

phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) was shown in

Arabidopsis in cold [100] NO appeared as a negative regulator of sphingolipid signalling as

enhanced NO impaired sphingolipids phosphorylation suggesting the fine-tuning of phospho-

versus unphosphorylated sphingolipid ratio probably by NO modifications As sphingolipid

kinasesphosphatases are not metal-dependent enzymes the possibility of the regulation by

metal nitrosation was ruled out [100] Tyrosine nitration might competitively inhibit tyrosine

phosphorylation As peroxinitrite promotes tyrosine nitration in cold a direct competition

between nitration and phosphorylation is predicted indicating a probable cross-talk between

these two PTMs This opens up a new area of research where PTM cross-talk in stress

conditions needs to be understood to dissect the fine regulatory circuitry of cold and NO

signalling

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

e8 Conclusions and Future Prospective

Overall it is clear that cold stress led to NO evolution in plants by both oxidative and

reductive pathways of NO production Enhanced NO in turn modulates various target

molecules including proteins and lipids As some proteins are the TFs regulation of these by

NO based PTMs in turns modulate the gene expression To summarize potential NO

signalling in cold stress a hypothetical model is proposed (Figure 4) The cellular membrane

being the outermost communication channel of the plant cell perceives cold stress signal

Cold stress causes loss of membrane integrity solute leakage and membrane rigidification

Cytoskeleton elements actin and microtubules act as low temperature sensors and activate

Ca2+

channels Increased Ca2+

level activate diacylglycerol kinase (DGK) DGK

phosphorylates diacylglycerol (DAG) and contributes to phosphatadic acid accumulation

Phosphatidic acid accumulation is an early response to cold stress As NO production is also

an early response to the cold stress its contribution in upstream events in cold signal

transduction is expected NO appears to compete with spingolipid phosphorylation by NO

based PTMs and might play an important role in the fine-tuning of phospho- versus

unphosphorylated sphingolipid ratio NO also promotes proline synthesis by cGMP

dependent signalling Cold stress causes an oxidative burst during which ROS are generated

Enhanced NO acts a ROS scavenger by 1) enhancing antioxidant enzymes (MDHAR

DHAR APX POD GR SOD and CAT) and antioxidant (ascorbate and GSH) accumulation

(2) reacting with superoxide radicals to produce peroxynitrite which in turn enhances protein

tyrosine nitration Cold induced SNOs increases S-nitrosylation of photosynthetic metabolic

defense and signaling related proteins Increased GSNOR activity suggested the activation of

regulatory mechanism for excessive removal of RNS in cold In addition cold tolerant plants

also reprogram their transcriptome in response to stress S-nitrosylation of the transcription

factors (WRKY TGA1 and NPR1) along with other nuclear proteins (histone deactylase and

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

eGAPDH) suggested NO dependent transcriptional regulation NO positively modulate the

expression of transcription factors (CBF1 and CBF3) and genes (COR15a LT130 and LT

178) components of ABA-dependent cold signalling pathway SIZ1 is tyrosine nitrated while

CBF2 and ZAT12 are unaffected by NO The regulation of ICE HOS1 and MYB15 by NO is

not known NO also seems to participate in the ABA-dependent cold signaling pathway as

DNA binding of MYB2 TF is negatively regulated by S-nitrosylation Thus indicating the

modulation of both ABA-dependent and ABA-independent pathways of cold stress signalling

by NO From the present model it can be predicted that the NO accumulated during cold

stress is likely to play an important role in orchestrating cellular responses by involving

multiple signaling pathways thus contributing to cold acclimation In addition exhaustive

analysis of cold responsive proteins for NO based PTMs identified HSPs GST DHAR

RuBisCO and GAPDH as S-nitrosylated S-glutathionylated and nitrated target and suggested

a probable cross-talk between these PTMs in stress Exploring the organelle proteome further

strengthened the role of NO signalling in spatial regulation of defencestress sugar

metabolism nuclear and cytoskeletal proteins Taken together existence of a novel

regulatory mechanism is proposed for NO in cold stress signaling Further knowledge

regarding the regulatory role of NO based PTMs in cold stress signalling cascade would be of

much interest In addition identification of in vivo NO modulated targets would further help

in exploring the complicated network of NO and cold stress signalling coss-talk

Acknowledgement

The described work is partially supported by the Special Assistant Programme (SAP F 3-

102011 SAP II) UGC grant to the Department of Botany a research grant provided by

University of Delhi and DBT grant (BTPR10799NDB511712008) from Department of

Biotechnology Government of India to RD

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

eThe authors have declared no conflict of interest

8 References

[1] Cramer G R Urano K Delrot S Pezzotti M Shinozaki K Effects of abiotic stress

on plants a systems biology perspective BMC Plant Biol 2011 11 163

[2] Petoukhov P Semenov V A A link between reduced Barents‐Kara sea ice and cold

winter extremes over northern continents J Geophys Res 2010 115 D21111

doi1010292009JD013568

[3] Thomashow M F Plant cold acclimation freezing tolerance genes and regulatory

mechanisms Annu Rev Plant Physiol Plant Mol Biol 1999 50 571ndash599

[4] Chinnusamy V Zhu J K Sunkar R Regulation During Cold Stress Acclimation in

Plants Methods Mol Biol 2010 639 39ndash55

[5] Siddiqui M H Al-Whaibi M H Basalah M O Role of nitric oxide in tolerance of

plants to abiotic stress Protoplasma 2011 248 447ndash455

[6] Gupta K J Hincha D K Mur L A NO way to treat a cold New Phytol 2011 189

360ndash363

[7] Abat J K Deswal R Differential modulation of S-nitrosoproteome of Brassica juncea

by low temperature change in S-nitrosylation of Rubisco is responsible for the inactivation

of its carboxylase activity Proteomics 2009 9 4368ndash4380

[8] Corpas F J Chaki M Fernaacutendez-Ocana A Valderrama R Metabolism of reactive

nitrogen species in pea plants under abiotic stress conditions Plant Cell Physiol 2008 49

1711ndash1722

[9] Airaki M Leterrier M Mateos R M Valderrama R et al Metabolism of reactive

oxygen species and reactive nitrogen species in pepper (Capsicum annuum L) plants under

low temperature stress Plant Cell Environ 2012 35 281ndash295

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

e[10] Gupta K J Fernie A R Kaiser W M van Dongen J T On the origins of nitric

oxide Trends Plant Sci 2011 16 160-168

[11] Liu Y Jiang H Zhao Z An L Nitric oxide synthase like activity-dependent nitric

oxide production protects against chilling-induced oxidative damage in Chorispora bungeana

suspension cultured cells Plant Physiol Biochem 2010 48 936ndash944

[12] Talwar P S Gupta R Maurya A K Deswal R Brassica juncea nitric oxide

synthase like activity is stimulated by PKC activators and calcium suggesting modulation by

PKC-like kinase Plant Physiol Biochem 2012 60 157ndash164

[13] Zhao R Sheng J Shengnan L V Zheng Y Nitric oxide participates in the

regulation of LeCBF1 gene expression and improves cold tolerance in harvested tomato fruit

Postharvest Biol Technol 2011 62 121ndash126

[14] Cantrel C Vazquez T Puyaubert J Rezeacute N et al Nitric oxide participates in cold-

responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana New

Phytol 2011 189 415ndash427

[15] Zhao M G Chen L Zhang L L Zhang W H Nitric reductase-dependent nitric

oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis Plant

Physiol 2009 151 755ndash767

[16] Bai X G Chen J H Kong X X Todd C D et al Carbon monoxide enhances the

chilling tolerance of recalcitrant Baccaurea ramiflora seeds via nitric oxide-mediated

glutathione homeostasis Free Radic Biol Med 2012 15 710-720

[17] Wang Y H Li X C Zhu-Ge Q Jiang X et al Nitric oxide participates in cold-

inhibited Camellia sinensis pollen germination and tube growth pvia cGMP in vitro PLoS

One 2012 7 doi 101371journalpone0052436

[18] Astiera J Rasula S Koena E Manzoora H et al S-nitrosylation An emerging post-

translational protein modification in plants Plant Sci 2011 181 527ndash533

Acc

epte

d A

rticl

e[19] Neill S J Desikan R Hancock J T Nitric oxide signalling in plants New Phytol

2003 159 11-35

[20] Saibo N J M Lourenco T Oliveira M M Transcription factors and regulation of

photosynthetic and related metabolism under environmental stresses Annal Bot 2009 103

609ndash623

[21] Miura K Jin J B Lee J Yoo C Y SIZ1-mediated sumoylation of ICE1 controls

CBF3DREB1A expression and freezing tolerance in Arabidopsis Plant Cell 2007 19

1403ndash1414

[22] Chinnusamy V Schumaker K Zhu J K Molecular genetic perspectives on cross-

talk and specificity in abiotic stress signalling in plants J Exp Bot 2003 55 225ndash236

[23] Dong C H Agarwal M Zhang Y Xie Q Zhu J K The negative regulator of

plant cold responses HOS1 is a RING E3 ligase that mediates the ubiquitination and

degradation of ICE1 Proc Natl Acad Sci U S A 2006 103 8281ndash8286

[24] Lozano-Juste J Colom-Moreno R Leoacuten J In vivo protein tyrosine nitration in

Arabidopsis thaliana J Exp Bot 2011 62 3501ndash3517

[25] Zhou M Q Shen C Wu L H Tang K X et al CBF-dependent signaling pathway

a key responder to low temperature stress in plants Crit Rev Biotechnol 2011 31 186-192

[26] Perazzolli M Dominici P Romero-Puertas M C Zago E Arabidopsis

nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity Plant Cell 2004 16

2785ndash2794

[27] Serpa V Vernal J Lamattina L Grotewold E et al Inhibition of AtMYB2 DNA-

binding by nitric oxide involves cysteine S-nitrosylation Biochem Biophys Res Commun

2007 361 1048ndash1053

[28] Foster M W Methodologies for the characterization identification and quantification

of S-nitrosylated proteins Biochim Biophys Acta 2012 1820 675-683

Acc

epte

d A

rticl

e[29] Raju K Doulias P T Tenopoulou M Greene J L Ischiropoulos H Strategies and

tools to explore protein S-nitrosylation Biochim Biophys Acta 2012 1820 684ndash688

[30] Gao X H Bedhomme M Veyel D Zaffagnini M Lemairem SD Methods for

analysis of protein glutathionylation and their application to photosynthetic organisms Mol

Plant 2009 2 218ndash235

[31] Sehrawat A Deswal R Protein Tyrosine Nitration in Abiotic Stress in Plants Plant

Stress (In Press)

[32] Jaffrey S R Snyder S H The biotin switch method for the detection of S-

nitrosylated proteins Sci STKE 2001 12 pl1

[33] Hao G Derakhshan B Shi L Campagne F et al SNOSID a proteomic method for

identification of cysteine S-nitrosylation sites in complex protein mixtures Proc Natl Acad

Sci U S A 2006 103 1012-1017

[34] Camerini S Polci M L Restuccia U Usuelli V et al A novel approach to identify

proteins modified by nitric oxide the HIS-TAG switch method J Proteome Res 2007 68

3224-3231

[35] Kettenhofen N J Wang X Gladwin M T Hogg N In-gel detection of S-nitrosated

proteins using fluorescence methods Methods Enzymol 2008 441 53ndash71

[36] Han P Zhou X Huang B Zhang X et al On-gel fluorescent visualization and the

site identification of S-nitrosylated proteins Anal Biochem 2008 377 150ndash155

[37] Forrester M T Thompson J W Foster M W Nogueira L et al Proteomic

analysis of S-nitrosylation and denitrosylation by resin-assisted capture Nat Biotechnol

2009 27 557-559

[38] Sinha V Wijewickrama G T Chandrasena R E Xu H et al Proteomic and mass

spectroscopic quantitation of protein S-nitrosation differentiates NO-donors ACS Chem Biol

2010 5 667ndash680

Acc

epte

d A

rticl

e[29] Raju K Doulias P T Tenopoulou M Greene J L Ischiropoulos H Strategies and

tools to explore protein S-nitrosylation Biochim Biophys Acta 2012 1820 684ndash688

[30] Gao X H Bedhomme M Veyel D Zaffagnini M Lemairem SD Methods for

analysis of protein glutathionylation and their application to photosynthetic organisms Mol

Plant 2009 2 218ndash235

[31] Sehrawat A Deswal R Protein Tyrosine Nitration in Abiotic Stress in Plants Plant

Stress (In Press)

[32] Jaffrey S R Snyder S H The biotin switch method for the detection of S-

nitrosylated proteins Sci STKE 2001 12 pl1

[33] Hao G Derakhshan B Shi L Campagne F et al SNOSID a proteomic method for

identification of cysteine S-nitrosylation sites in complex protein mixtures Proc Natl Acad

Sci U S A 2006 103 1012-1017

[34] Camerini S Polci M L Restuccia U Usuelli V et al A novel approach to identify

proteins modified by nitric oxide the HIS-TAG switch method J Proteome Res 2007 68

3224-3231

[35] Kettenhofen N J Wang X Gladwin M T Hogg N In-gel detection of S-nitrosated

proteins using fluorescence methods Methods Enzymol 2008 441 53ndash71

[36] Han P Zhou X Huang B Zhang X et al On-gel fluorescent visualization and the

site identification of S-nitrosylated proteins Anal Biochem 2008 377 150ndash155

[37] Forrester M T Thompson J W Foster M W Nogueira L et al Proteomic

analysis of S-nitrosylation and denitrosylation by resin-assisted capture Nat Biotechnol

2009 27 557-559

[38] Sinha V Wijewickrama G T Chandrasena R E Xu H et al Proteomic and mass

spectroscopic quantitation of protein S-nitrosation differentiates NO-donors ACS Chem Biol

2010 5 667ndash680

Acc

epte

d A

rticl

e[39] Faccenda A Bonham C A Vacratsis P O Zhang X et al Gold nanoparticle

enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins J

Am Chem Soc 2010 132 11392-11394

[40] Zhang J Li S Zhang D Wang H et al Reductive ligation mediated one-step

disulfide formation of S-nitrosothiols Org Lett 2010 12 4208-4211

[41] Doulias P T Greene J L Greco T M Tenopoulou M et al Structural profiling of

endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse

mechanisms for protein S-nitrosylation Proc Natl Acad Sci U S A 2010 107 16958-

16963

[42] Michelet L Zaffagnini M Vanacker H Le Mareacutechal et al In vivo targets of S-

thiolation in Chlamydomonas reinhardtii J Biol Chem 2008 283 21571ndash21580

[43] Dixon D P Skipsey M Grundy N M Edwards R Stress-induced protein S-

glutathionylation in Arabidopsis Plant Physiol 2005 138 2233ndash2244

[44] Lind C Gerdes R Hamnell Y Schuppe-Koistinen I et al Identification of S-

glutathionylated cellular proteins during oxidative stress and constitutive metabolism by

affinity purification and proteomic analysis Arch Biochem Biophys 2002 406 229-240

[45] Cheng G Ikeda Y Iuchi Y Fujii J Detection of S-glutathionylated proteins by

glutathione S-transferase overlay Arch Biochem Biophys 2005 435 42ndash49

[46] Chaki M Valderrama R Fernaacutendez-Ocantildea A M Carreras A et al High

temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of

nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine

nitration Plant Cell Environ 2011 34 1803ndash1818

[47] Larsen T R Soumlderling A S Caidahl K Roepstorff P et al Nitration of soluble

proteins in organotypic culture models of Parkinsons disease Neurochem Int 2008 52487-

494

Acc

epte

d A

rticl

e[48] Larsen T R Bache N Gramsbergen J B Roepstorff P Identification of

nitrotyrosine containing peptides using combined fractional diagonal chromatography

(COFRADIC) and off-line nano-LC-MALDI J Am Soc Mass Spectrom 2011 22 989-

996

[49] Neilson K A Mariani M Haynes P A Quantitative proteomic analysis of cold-

responsive proteins in rice Proteomics 2011 11 1696ndash1706

[50] Tanou G Filippou P Belghazi M Job D et al Oxidative and nitrosative-based

signaling and associated post-translational modifications orchestrate the acclimation of citrus

plants to salinity stress Plant J 2012 doi101111j1365-313X201205100

[51] Lee D G Ahsan N Lee S H Lee J J et al Chilling stress-induced proteomic

changes in rice roots J Plant Physiol 2009 166 1ndash11

[52] Hashimoto M Komatsu S Proteomics analysis of rice seedling during cold stress

Proteomics 2007 7 1293ndash1302

[53] Degand H Faber A M Dauchot N Mingeot D et al Proteomic analysis of chicory

root identifies proteins typically involved in cold acclimation Proteomics 2009 9 2903ndash

2907

[54] Uvaacutečkovaacute L Takaacuteč T Boehm N Obert B Šamaj J Proteomic and biochemical

analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an

important role of anti-oxidative enzymes J Proteomics 2012 75 1886ndash1894

[55] Imin N Kerim T Weinman J J Rolfe B G Low temperature treatment at the

young microspore stage induces protein changes in rice anthers Mol Cell Proteomics 2006

5 274ndash292

[56] Imin N Kerim T Rolfe B G Weinman J J Effect of early cold stress on the

maturation of rice anthers Proteomics 2004 4 1873ndash1882

Acc

epte

d A

rticl

e[57] Cheng L Gao X Li S Shi M et al Proteomic analysis of soybean [Glycine max

(L) Meer] seeds during imbibition at chilling temperature Mol Breed 2010 26 1ndash17

[58] Zheng M Wang Y Liu K Shu H Zhou Z Protein expression changes during

cotton fiber elongation in response to low temperature stress J Plant Physiol 2012 169

399ndash409

[59] Reyes‐Dıaz M Ulloa N Zuniga‐Feest A Gutierrez A et al Arabidopsis thaliana

avoids freezing by supercooling J Exp Bot 2006 57 3687ndash3696

[60] Bogdanovic J Mojovic M Milosavic N Mitrovic A et al Role of fructose in the

adaptation of plants to cold induced oxidative stress Euro Biophys J 2008 37 1241ndash1246

[61] Kawamura Y Uemura M Mass spectrometric approach for identifying putative

plasma membrane proteins of Arabidopsis leaves associated with cold acclimation Plant J

2003 36 141ndash154

[62] Goulas E Schubert M Kieselbach T Kleczkowski A L et al The chloroplast

lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short-

and long-term exposure to low temperature Plant J 2006 47 720ndash734

[63] Yan S P Zhang Q Y Tang Z C Su W A Sun W N Comparative proteomic

analysis provides new insights into chilling stress responses in rice Mol Cell Proteomics

2006 5 484ndash96

[64] Cui S Huang F Wang J Ma X et al A proteomic analysis of cold stress responses

in rice seedlings Proteomics 2005 5 3162ndash3172

[65] Fares A Rossignol M Peltier J B Proteomics investigation of endogenous S-

nitrosylation in Arabidopsis Biochem Biophys Res Commun 2011 416 331ndash336

[66] Amme S Matros A Schlesier B Mock H P Proteome analysis of cold stress

response in Arabidopsis thaliana using DIGE-technology J Exp Bot 2006 57 1537ndash1546

Acc

epte

d A

rticl

e[67] Balbuena T S Salas J J Martiacutenez-Force E Garces R Thelen J J Proteome

analysis of cold acclimation in sunflower J Proteome Res 2011 10 2330ndash2346

[68] Sarhadi E Mahfoozi S Hosseini S A Salekdeh G H Cold acclimation proteome

analysis reveals close link between the up-regulation of low-temperature associated proteins

and vernalization fulfilment J Proteome Res 2010 9 5658ndash5667

[69] Gupta R Deswal R Low temperature stress modulated secretome analysis and

purification of antifreeze protein from Hippophae rhamnoides a Himalayan wonder plant J

Proteome Res 2012 11 2684ndash2696

[70] Hashimoto M Toorchi M Matsushita K Iwasaki Y Komatsu S Proteome

analysis of rice root plasma membrane and detection of cold stress responsive proteins

Protein Peptide Lett 2009 16 685ndash697

[71] Nilo R Saffie C Lilley K Baeza-Yates R et al Proteomic analysis of peach fruit

mesocarp softening and chilling injury using difference gel electrophoresis (DIGE) BMC

Genomics 2010 11 43

[72] Chattopadhyay A Subba P Pandey A Bhushan D et al Analysis of the grasspea

proteome and identification of stress-responsive proteins upon exposure to high salinity low

temperature and abscisic acid treatment Phytochemistry 2011 72 1293ndash1307

[73] Yun Z Jin S Ding Y Wang Z et al Comparative transcriptomics and proteomics

analysis of citrus fruit to improve understanding of the effect of low temperature on

maintaining fruit quality during lengthy post-harvest storage J Exp Bot 2012 63 2873ndash

2893

[74] Herman E M Rotter K Premakumar R Elwinger G et al Additional freeze

hardiness in wheat acquired by exposure to -3 ordmC is associated with extensive physiological

morphological and molecular changes J Exp Bot 2006 14 3601ndash3618

Acc

epte

d A

rticl

e[75] Kato H Takemoto D Kawakita K Proteomic analysis of S-nitrosylated proteins in

potato plant Physiol Plant 2012 doi 101111j1399-3054201201684x

[76] Maldonado-Alconada A M Zomeno S E Lindermayr C Redondo-Lopez I et al

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with

Pseudomonas syringae Acta Physiol Plant 2010 33 1493ndash1514

[77] Lin A Wang Y Tang J Xue P et al Nitric oxide and protein S-nitrosylation are

integral to hydrogen peroxide-induced leaf cell death in rice Plant Physiol 2012 158 451ndash

464

[78] Viacutetaacutemvaacutes P Praacutešil I T Kosova K Planchon S Renaut J Analysis of proteome and

frost tolerance in chromosome 5A and 5B reciprocal substitution lines between two winter

wheats during long-term cold acclimation J Proteomics 2012 12 68ndash85

[79] Bravo L A Gallardo J Navarrete A Olave N et al Cryoprotective activity of a

cold induced dehydrin purified from barley Physiol Plant 2003 118 262ndash269

[80] Wisniewski M Webb R Balsamo R Close T J et al Purification

immunolocalization cryoprotective and antifreeze activity of PCA60 A dehydrin from peach

(Prunus persica) Physiol Plant 1999 105 600ndash608

[81] Simpson D J Smallwood M Twigg S Doucet C J et al Purification and

characterisation of an antifreeze protein from Forsythia suspensa (L) Cryobiology 2005 51

230ndash234

[82] Hara M Fujinaga M and Kuboi T Radical scavenging activity and oxidative

modification of citrus dehydrin Plant Physiol Biochem 2004 42 657ndash862

[83] Lindermayr C Saalbach G Durner J Proteomic identification of S-nitrosylated

proteins in Arabidopsis Plant Physiol 2005 137 921ndash930

Acc

epte

d A

rticl

e[48] Larsen T R Bache N Gramsbergen J B Roepstorff P Identification of

nitrotyrosine containing peptides using combined fractional diagonal chromatography

(COFRADIC) and off-line nano-LC-MALDI J Am Soc Mass Spectrom 2011 22 989-

996

[49] Neilson K A Mariani M Haynes P A Quantitative proteomic analysis of cold-

responsive proteins in rice Proteomics 2011 11 1696ndash1706

[50] Tanou G Filippou P Belghazi M Job D et al Oxidative and nitrosative-based

signaling and associated post-translational modifications orchestrate the acclimation of citrus

plants to salinity stress Plant J 2012 doi101111j1365-313X201205100

[51] Lee D G Ahsan N Lee S H Lee J J et al Chilling stress-induced proteomic

changes in rice roots J Plant Physiol 2009 166 1ndash11

[52] Hashimoto M Komatsu S Proteomics analysis of rice seedling during cold stress

Proteomics 2007 7 1293ndash1302

[53] Degand H Faber A M Dauchot N Mingeot D et al Proteomic analysis of chicory

root identifies proteins typically involved in cold acclimation Proteomics 2009 9 2903ndash

2907

[54] Uvaacutečkovaacute L Takaacuteč T Boehm N Obert B Šamaj J Proteomic and biochemical

analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an

important role of anti-oxidative enzymes J Proteomics 2012 75 1886ndash1894

[55] Imin N Kerim T Weinman J J Rolfe B G Low temperature treatment at the

young microspore stage induces protein changes in rice anthers Mol Cell Proteomics 2006

5 274ndash292

[56] Imin N Kerim T Rolfe B G Weinman J J Effect of early cold stress on the

maturation of rice anthers Proteomics 2004 4 1873ndash1882

Acc

epte

d A

rticl

e[57] Cheng L Gao X Li S Shi M et al Proteomic analysis of soybean [Glycine max

(L) Meer] seeds during imbibition at chilling temperature Mol Breed 2010 26 1ndash17

[58] Zheng M Wang Y Liu K Shu H Zhou Z Protein expression changes during

cotton fiber elongation in response to low temperature stress J Plant Physiol 2012 169

399ndash409

[59] Reyes‐Dıaz M Ulloa N Zuniga‐Feest A Gutierrez A et al Arabidopsis thaliana

avoids freezing by supercooling J Exp Bot 2006 57 3687ndash3696

[60] Bogdanovic J Mojovic M Milosavic N Mitrovic A et al Role of fructose in the

adaptation of plants to cold induced oxidative stress Euro Biophys J 2008 37 1241ndash1246

[61] Kawamura Y Uemura M Mass spectrometric approach for identifying putative

plasma membrane proteins of Arabidopsis leaves associated with cold acclimation Plant J

2003 36 141ndash154

[62] Goulas E Schubert M Kieselbach T Kleczkowski A L et al The chloroplast

lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short-

and long-term exposure to low temperature Plant J 2006 47 720ndash734

[63] Yan S P Zhang Q Y Tang Z C Su W A Sun W N Comparative proteomic

analysis provides new insights into chilling stress responses in rice Mol Cell Proteomics

2006 5 484ndash96

[64] Cui S Huang F Wang J Ma X et al A proteomic analysis of cold stress responses

in rice seedlings Proteomics 2005 5 3162ndash3172

[65] Fares A Rossignol M Peltier J B Proteomics investigation of endogenous S-

nitrosylation in Arabidopsis Biochem Biophys Res Commun 2011 416 331ndash336

[66] Amme S Matros A Schlesier B Mock H P Proteome analysis of cold stress

response in Arabidopsis thaliana using DIGE-technology J Exp Bot 2006 57 1537ndash1546

Acc

epte

d A

rticl

e[67] Balbuena T S Salas J J Martiacutenez-Force E Garces R Thelen J J Proteome

analysis of cold acclimation in sunflower J Proteome Res 2011 10 2330ndash2346

[68] Sarhadi E Mahfoozi S Hosseini S A Salekdeh G H Cold acclimation proteome

analysis reveals close link between the up-regulation of low-temperature associated proteins

and vernalization fulfilment J Proteome Res 2010 9 5658ndash5667

[69] Gupta R Deswal R Low temperature stress modulated secretome analysis and

purification of antifreeze protein from Hippophae rhamnoides a Himalayan wonder plant J

Proteome Res 2012 11 2684ndash2696

[70] Hashimoto M Toorchi M Matsushita K Iwasaki Y Komatsu S Proteome

analysis of rice root plasma membrane and detection of cold stress responsive proteins

Protein Peptide Lett 2009 16 685ndash697

[71] Nilo R Saffie C Lilley K Baeza-Yates R et al Proteomic analysis of peach fruit

mesocarp softening and chilling injury using difference gel electrophoresis (DIGE) BMC

Genomics 2010 11 43

[72] Chattopadhyay A Subba P Pandey A Bhushan D et al Analysis of the grasspea

proteome and identification of stress-responsive proteins upon exposure to high salinity low

temperature and abscisic acid treatment Phytochemistry 2011 72 1293ndash1307

[73] Yun Z Jin S Ding Y Wang Z et al Comparative transcriptomics and proteomics

analysis of citrus fruit to improve understanding of the effect of low temperature on

maintaining fruit quality during lengthy post-harvest storage J Exp Bot 2012 63 2873ndash

2893

[74] Herman E M Rotter K Premakumar R Elwinger G et al Additional freeze

hardiness in wheat acquired by exposure to -3 ordmC is associated with extensive physiological

morphological and molecular changes J Exp Bot 2006 14 3601ndash3618

Acc

epte

d A

rticl

e[75] Kato H Takemoto D Kawakita K Proteomic analysis of S-nitrosylated proteins in

potato plant Physiol Plant 2012 doi 101111j1399-3054201201684x

[76] Maldonado-Alconada A M Zomeno S E Lindermayr C Redondo-Lopez I et al

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with

Pseudomonas syringae Acta Physiol Plant 2010 33 1493ndash1514

[77] Lin A Wang Y Tang J Xue P et al Nitric oxide and protein S-nitrosylation are

integral to hydrogen peroxide-induced leaf cell death in rice Plant Physiol 2012 158 451ndash

464

[78] Viacutetaacutemvaacutes P Praacutešil I T Kosova K Planchon S Renaut J Analysis of proteome and

frost tolerance in chromosome 5A and 5B reciprocal substitution lines between two winter

wheats during long-term cold acclimation J Proteomics 2012 12 68ndash85

[79] Bravo L A Gallardo J Navarrete A Olave N et al Cryoprotective activity of a

cold induced dehydrin purified from barley Physiol Plant 2003 118 262ndash269

[80] Wisniewski M Webb R Balsamo R Close T J et al Purification

immunolocalization cryoprotective and antifreeze activity of PCA60 A dehydrin from peach

(Prunus persica) Physiol Plant 1999 105 600ndash608

[81] Simpson D J Smallwood M Twigg S Doucet C J et al Purification and

characterisation of an antifreeze protein from Forsythia suspensa (L) Cryobiology 2005 51

230ndash234

[82] Hara M Fujinaga M and Kuboi T Radical scavenging activity and oxidative

modification of citrus dehydrin Plant Physiol Biochem 2004 42 657ndash862

[83] Lindermayr C Saalbach G Durner J Proteomic identification of S-nitrosylated

proteins in Arabidopsis Plant Physiol 2005 137 921ndash930

Acc

epte

d A

rticl

e[57] Cheng L Gao X Li S Shi M et al Proteomic analysis of soybean [Glycine max

(L) Meer] seeds during imbibition at chilling temperature Mol Breed 2010 26 1ndash17

[58] Zheng M Wang Y Liu K Shu H Zhou Z Protein expression changes during

cotton fiber elongation in response to low temperature stress J Plant Physiol 2012 169

399ndash409

[59] Reyes‐Dıaz M Ulloa N Zuniga‐Feest A Gutierrez A et al Arabidopsis thaliana

avoids freezing by supercooling J Exp Bot 2006 57 3687ndash3696

[60] Bogdanovic J Mojovic M Milosavic N Mitrovic A et al Role of fructose in the

adaptation of plants to cold induced oxidative stress Euro Biophys J 2008 37 1241ndash1246

[61] Kawamura Y Uemura M Mass spectrometric approach for identifying putative

plasma membrane proteins of Arabidopsis leaves associated with cold acclimation Plant J

2003 36 141ndash154

[62] Goulas E Schubert M Kieselbach T Kleczkowski A L et al The chloroplast

lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short-

and long-term exposure to low temperature Plant J 2006 47 720ndash734

[63] Yan S P Zhang Q Y Tang Z C Su W A Sun W N Comparative proteomic

analysis provides new insights into chilling stress responses in rice Mol Cell Proteomics

2006 5 484ndash96

[64] Cui S Huang F Wang J Ma X et al A proteomic analysis of cold stress responses

in rice seedlings Proteomics 2005 5 3162ndash3172

[65] Fares A Rossignol M Peltier J B Proteomics investigation of endogenous S-

nitrosylation in Arabidopsis Biochem Biophys Res Commun 2011 416 331ndash336

[66] Amme S Matros A Schlesier B Mock H P Proteome analysis of cold stress

response in Arabidopsis thaliana using DIGE-technology J Exp Bot 2006 57 1537ndash1546

Acc

epte

d A

rticl

e[67] Balbuena T S Salas J J Martiacutenez-Force E Garces R Thelen J J Proteome

analysis of cold acclimation in sunflower J Proteome Res 2011 10 2330ndash2346

[68] Sarhadi E Mahfoozi S Hosseini S A Salekdeh G H Cold acclimation proteome

analysis reveals close link between the up-regulation of low-temperature associated proteins

and vernalization fulfilment J Proteome Res 2010 9 5658ndash5667

[69] Gupta R Deswal R Low temperature stress modulated secretome analysis and

purification of antifreeze protein from Hippophae rhamnoides a Himalayan wonder plant J

Proteome Res 2012 11 2684ndash2696

[70] Hashimoto M Toorchi M Matsushita K Iwasaki Y Komatsu S Proteome

analysis of rice root plasma membrane and detection of cold stress responsive proteins

Protein Peptide Lett 2009 16 685ndash697

[71] Nilo R Saffie C Lilley K Baeza-Yates R et al Proteomic analysis of peach fruit

mesocarp softening and chilling injury using difference gel electrophoresis (DIGE) BMC

Genomics 2010 11 43

[72] Chattopadhyay A Subba P Pandey A Bhushan D et al Analysis of the grasspea

proteome and identification of stress-responsive proteins upon exposure to high salinity low

temperature and abscisic acid treatment Phytochemistry 2011 72 1293ndash1307

[73] Yun Z Jin S Ding Y Wang Z et al Comparative transcriptomics and proteomics

analysis of citrus fruit to improve understanding of the effect of low temperature on

maintaining fruit quality during lengthy post-harvest storage J Exp Bot 2012 63 2873ndash

2893

[74] Herman E M Rotter K Premakumar R Elwinger G et al Additional freeze

hardiness in wheat acquired by exposure to -3 ordmC is associated with extensive physiological

morphological and molecular changes J Exp Bot 2006 14 3601ndash3618

Acc

epte

d A

rticl

e[75] Kato H Takemoto D Kawakita K Proteomic analysis of S-nitrosylated proteins in

potato plant Physiol Plant 2012 doi 101111j1399-3054201201684x

[76] Maldonado-Alconada A M Zomeno S E Lindermayr C Redondo-Lopez I et al

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with

Pseudomonas syringae Acta Physiol Plant 2010 33 1493ndash1514

[77] Lin A Wang Y Tang J Xue P et al Nitric oxide and protein S-nitrosylation are

integral to hydrogen peroxide-induced leaf cell death in rice Plant Physiol 2012 158 451ndash

464

[78] Viacutetaacutemvaacutes P Praacutešil I T Kosova K Planchon S Renaut J Analysis of proteome and

frost tolerance in chromosome 5A and 5B reciprocal substitution lines between two winter

wheats during long-term cold acclimation J Proteomics 2012 12 68ndash85

[79] Bravo L A Gallardo J Navarrete A Olave N et al Cryoprotective activity of a

cold induced dehydrin purified from barley Physiol Plant 2003 118 262ndash269

[80] Wisniewski M Webb R Balsamo R Close T J et al Purification

immunolocalization cryoprotective and antifreeze activity of PCA60 A dehydrin from peach

(Prunus persica) Physiol Plant 1999 105 600ndash608

[81] Simpson D J Smallwood M Twigg S Doucet C J et al Purification and

characterisation of an antifreeze protein from Forsythia suspensa (L) Cryobiology 2005 51

230ndash234

[82] Hara M Fujinaga M and Kuboi T Radical scavenging activity and oxidative

modification of citrus dehydrin Plant Physiol Biochem 2004 42 657ndash862

[83] Lindermayr C Saalbach G Durner J Proteomic identification of S-nitrosylated

proteins in Arabidopsis Plant Physiol 2005 137 921ndash930

Acc

epte

d A

rticl

e[67] Balbuena T S Salas J J Martiacutenez-Force E Garces R Thelen J J Proteome

analysis of cold acclimation in sunflower J Proteome Res 2011 10 2330ndash2346

[68] Sarhadi E Mahfoozi S Hosseini S A Salekdeh G H Cold acclimation proteome

analysis reveals close link between the up-regulation of low-temperature associated proteins

and vernalization fulfilment J Proteome Res 2010 9 5658ndash5667

[69] Gupta R Deswal R Low temperature stress modulated secretome analysis and

purification of antifreeze protein from Hippophae rhamnoides a Himalayan wonder plant J

Proteome Res 2012 11 2684ndash2696

[70] Hashimoto M Toorchi M Matsushita K Iwasaki Y Komatsu S Proteome

analysis of rice root plasma membrane and detection of cold stress responsive proteins

Protein Peptide Lett 2009 16 685ndash697

[71] Nilo R Saffie C Lilley K Baeza-Yates R et al Proteomic analysis of peach fruit

mesocarp softening and chilling injury using difference gel electrophoresis (DIGE) BMC

Genomics 2010 11 43

[72] Chattopadhyay A Subba P Pandey A Bhushan D et al Analysis of the grasspea

proteome and identification of stress-responsive proteins upon exposure to high salinity low

temperature and abscisic acid treatment Phytochemistry 2011 72 1293ndash1307

[73] Yun Z Jin S Ding Y Wang Z et al Comparative transcriptomics and proteomics

analysis of citrus fruit to improve understanding of the effect of low temperature on

maintaining fruit quality during lengthy post-harvest storage J Exp Bot 2012 63 2873ndash

2893

[74] Herman E M Rotter K Premakumar R Elwinger G et al Additional freeze

hardiness in wheat acquired by exposure to -3 ordmC is associated with extensive physiological

morphological and molecular changes J Exp Bot 2006 14 3601ndash3618

Acc

epte

d A

rticl

e[75] Kato H Takemoto D Kawakita K Proteomic analysis of S-nitrosylated proteins in

potato plant Physiol Plant 2012 doi 101111j1399-3054201201684x

[76] Maldonado-Alconada A M Zomeno S E Lindermayr C Redondo-Lopez I et al

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with

Pseudomonas syringae Acta Physiol Plant 2010 33 1493ndash1514

[77] Lin A Wang Y Tang J Xue P et al Nitric oxide and protein S-nitrosylation are

integral to hydrogen peroxide-induced leaf cell death in rice Plant Physiol 2012 158 451ndash

464

[78] Viacutetaacutemvaacutes P Praacutešil I T Kosova K Planchon S Renaut J Analysis of proteome and

frost tolerance in chromosome 5A and 5B reciprocal substitution lines between two winter

wheats during long-term cold acclimation J Proteomics 2012 12 68ndash85

[79] Bravo L A Gallardo J Navarrete A Olave N et al Cryoprotective activity of a

cold induced dehydrin purified from barley Physiol Plant 2003 118 262ndash269

[80] Wisniewski M Webb R Balsamo R Close T J et al Purification

immunolocalization cryoprotective and antifreeze activity of PCA60 A dehydrin from peach

(Prunus persica) Physiol Plant 1999 105 600ndash608

[81] Simpson D J Smallwood M Twigg S Doucet C J et al Purification and

characterisation of an antifreeze protein from Forsythia suspensa (L) Cryobiology 2005 51

230ndash234

[82] Hara M Fujinaga M and Kuboi T Radical scavenging activity and oxidative

modification of citrus dehydrin Plant Physiol Biochem 2004 42 657ndash862

[83] Lindermayr C Saalbach G Durner J Proteomic identification of S-nitrosylated

proteins in Arabidopsis Plant Physiol 2005 137 921ndash930

Acc

epte

d A

rticl

e[75] Kato H Takemoto D Kawakita K Proteomic analysis of S-nitrosylated proteins in

potato plant Physiol Plant 2012 doi 101111j1399-3054201201684x

[76] Maldonado-Alconada A M Zomeno S E Lindermayr C Redondo-Lopez I et al

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with

Pseudomonas syringae Acta Physiol Plant 2010 33 1493ndash1514

[77] Lin A Wang Y Tang J Xue P et al Nitric oxide and protein S-nitrosylation are

integral to hydrogen peroxide-induced leaf cell death in rice Plant Physiol 2012 158 451ndash

464

[78] Viacutetaacutemvaacutes P Praacutešil I T Kosova K Planchon S Renaut J Analysis of proteome and

frost tolerance in chromosome 5A and 5B reciprocal substitution lines between two winter

wheats during long-term cold acclimation J Proteomics 2012 12 68ndash85

[79] Bravo L A Gallardo J Navarrete A Olave N et al Cryoprotective activity of a

cold induced dehydrin purified from barley Physiol Plant 2003 118 262ndash269

[80] Wisniewski M Webb R Balsamo R Close T J et al Purification

immunolocalization cryoprotective and antifreeze activity of PCA60 A dehydrin from peach

(Prunus persica) Physiol Plant 1999 105 600ndash608

[81] Simpson D J Smallwood M Twigg S Doucet C J et al Purification and

characterisation of an antifreeze protein from Forsythia suspensa (L) Cryobiology 2005 51

230ndash234

[82] Hara M Fujinaga M and Kuboi T Radical scavenging activity and oxidative

modification of citrus dehydrin Plant Physiol Biochem 2004 42 657ndash862

[83] Lindermayr C Saalbach G Durner J Proteomic identification of S-nitrosylated

proteins in Arabidopsis Plant Physiol 2005 137 921ndash930

Acc

epte

d A

rticl

e[84] Renaut J Hausman J F Bassett C Artlip T et al Quantitative proteomic analysis

of short photoperiod and low-temperature responses in bark tissues of peach (Prunus persica

L Batsch) Tree Genet Genomes 2008 4 589ndash600

[85] Wang X Yang P Zhang X Xu Y et al Proteomic analysis of the cold stress

response in the moss Physcomitrella patens Proteomics 2009 9 4529ndash4538

[86] Viktorova L V Maksyutova N N Trifonova T V Andrianov V V Production of

hydrogen peroxide and nitric oxide following introduction of nitrate and nitrite into wheat

leaf apoplast Biochemistry 2010 75 95ndash100

[87] Yemets A I Krasylenko Y A Lytvyn D I Sheremet Y A Blume Y B Nitric

oxide signalling via cytoskeleton in plants Plant Sci 2011 181 545ndash554

[88] Zaffagnini M Bedhomme M Groni H Marchand C H et al Glutathionylation in

the photosynthetic model organism Chlamydomonas reinhardtii a proteomic survey Mol

Cell Proteomics 2012 11 M111014142

[89] Michelet L Zaffagnini M Marchand C Collin V et al Glutathionylation of

chloroplast thioredoxin f is a redox signaling mechanism in plants Proc Natl Acad Sci U S

A 2005 102 16478ndash1683

[90] Taylor N L Heazlewood J L Day D A Millar A H Differential impact of

environmental stresses on the pea mitochondrial proteome Mol Cell Proteomics 2005 4

1122ndash1133

[91] Palmieri M C Lindermayr C Bauwe H Steinhauser C Durner J Regulation of

plant glycine decarboxylase by S-nitrosylation and glutathionylation Plant Physiol 2010

152 1514ndash1528

[92] Romero-Puertas M C Campostrini N Matte A Righetti P G et al Proteomic

analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive

response Proteomics 2008 8 1459ndash1469

Acc

epte

d A

rticl

e[93] Bae M S Cho E J Choi E Y Park O K Analysis of the Arabidopsis nuclear

proteome and its response to cold stress Plant J 2003 36 652ndash663

[94] Tada Y Spoel S H Pajerowska-Mukhtar K Mou Z et al Plant immunity requires

conformational changes [corrected] of NPR1 via S-nitrosylation and thioredoxins Science

2008 321 952ndash960

[95] Lindermayr C Sell S Muumlller B Leister D Durner J Redox regulation of the

NPR1-TGA1 system of Arabidopsis thaliana by nitric oxide Plant Cell 2010 22 2894ndash

2907

[96] Kornberg M D Sen N Hara M R Juluri K R et al GAPDH mediates

nitrosylation of nuclear proteins Nat Cell Biol 2010 12 1094ndash1100

[97] Hara M R Agrawal N Kim S F Cascio M B et al S-nitrosylated GAPDH

initiates apoptotic cell death by nuclear translocation following Siah1 binding Nat Cell Biol

2005 7 665ndash674

[98] Wawer I Bucholc M Astier J Anielska-Mazur A et al Regulation of Nicotiana

tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric

oxide in response to salinity Biochem J 2010 429 73ndash83

[99] Quilis J Pentildeas G Messeguer J Brugidou C San Segundo B The Arabidopsis

AtNPR1 inversely modulates defense responses against fungal bacterial or viral pathogens

while conferring hypersensitivity to abiotic stresses in transgenic rice Mol Plant Microbe

Interact 2008 21 1215ndash1231

[100] Guillas I Zachowski A Baudouin E A matter of fat interaction between nitric

oxide and sphingolipid signaling in plant cold response Plant Signal Behav 2011 6 140ndash

142

[101] Lee D G Ahsan N Lee S H Kang K Y et al An approach to identify cold-

induced low-abundant proteins in rice leaf C R Biol 2007a 330 215ndash225

Acc

epte

d A

rticl

e[102] Komatsu S Yamada E Furukawa K Cold stress changes the concanavalin A-

positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths

Amino Acids 2009 36 115ndash123

[103] Yang P F Li X J Liang Y Jing Y X et al Proteomic analysis of the response of

Liangyoupeijiu (Super High-Yield Hybrid Rice) seedlings to cold stress J Integr Plant Biol

2006 48 945minus951

[104] Rinalducci S Egidi M G Karimzadeh G Jazii F R Zolla L Proteomic analysis

of a spring wheat cultivar in response to prolonged cold stress Electrophoresis 2011 32

1807ndash1818

[105] Kosmala A Bocian A Rapacz M Jurczyk B Zwierzykowski Z Identification of

leaf proteins differentially accumulated during cold acclimation between Festuca pratensis

plants with distinct levels of frost tolerance J Exp Bot 2009 60 3595ndash3609

[106] Dumont E Bahrman N Goulas E Valot B et al A proteomic approach to

decipher chilling response from cold acclimation in pea (Pisum sativum L) Plant Sci 2011

180 86ndash98

[107] Gao F Zhou Y Zhu W Li X et al Proteomic analysis of cold stress-responsive

proteins in Thellungiella rosette leaves Planta 2009 230 1033ndash1046

[108] Jin Y Zhang C Yang H Yang Y et al Proteomic analysis of cold stress

responses in tobacco seedlings Afr J Biotechol 2011 10 18991ndash19004

[109] Evers D Legay S Lamoureux D Hausman J F et al Towards a synthetic view of

potato cold and salt stress response by transcriptomic and proteomic analyses Plant Mol

Biol 2012 78 503ndash514

[110] Long G Song J Deng Z Liu J Rao L Ptcorp gene induced by cold stress was

identified by proteomic analysis in leaves of Poncirus trifoliata (L) Raf Mol Biol Rep

2012 39 5859ndash5866

Acc

epte

d A

rticl

e[111] Renaut J Lutts S Hoffmann L Hausman J F Responses of poplar to chilling

temperatures proteomics and physiological aspects Plant Biol 2004 6 81ndash90

[112] Koehler G Wilson R C Goodpaster J V Soslashnsteby A et al Proteomic study of

low-temperature responses in strawberry cultivars (Fragaria times ananassa) that differ in cold

tolerance Plant Physiol 2012 159 1787ndash1805

[113] Kjellsen T D Shiryaeva L Schroumlderw P Strimbeck R Proteomics of extreme

freezing tolerance in Siberian spruce (Picea obovata) J Proteomics 2010 73 965ndash975

[114] Chen J Tian L Xu H Tian D et al Cold-induced changes of protein and

phosphoprotein expression patterns from rice roots as revealed by multiplex proteomic

analysis Plant Omics J 2012 5 194-199

[115] Gai Y P Ji X L Lu W Han X J et al A novel late embryogenesis abundant like

protein associated with chilling stress in Nicotiana tabacum cv bright yellow-2 cell

suspension culture Mol Cell Proteomics 2011 10 M111010363

[116] Gammulla C G Pascovici D Atwell B J Haynes P A Differential metabolic

response of cultured rice (Oryza sativa) cells exposed to high- and low-temperature stress

Proteomics 2010 10 3001ndash3019

[117] Sanchez-Bel P Egea I Sanchez-Ballesta M T Sevillano L Proteome changes in

tomato fruits prior to visible symptoms of chilling injury are linked to defensive mechanisms

uncoupling of photosynthetic processes and protein degradation machinery Plant Cell

Physiol 2012 53 470ndash484

[118] Tanou G Job C Rajjou L Arc E et al Proteomics reveals the overlapping roles

of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity Plant J

2009 60 795ndash804

Acc

epte

d A

rticl

e[119] Ortega-Galisteo A P Rodriacuteguez-Serrano M Pazmintildeo D M Gupta D K et al S-

Nitrosylated proteins in pea (Pisum sativum L) leaf peroxisomes changes under abiotic

stress J Exp Bot 2012 63 2089ndash2103

[120] Ito H Iwabuchi M Ogawa K The sugar-metabolic enzymes aldolase and triose-

phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana detection using

biotinylated glutathione Plant Cell Physiol 2003 44 655ndash660

[121] Zaffagnini M Michelet L Marchand C Sparla F et al The thioredoxin-

independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is

selectively regulated by glutathionylation FEBS J 2007 274 212ndash126

Figure Legends

Figure 1 Schematic diagram showing the methodology utilized for the identification of cold

stress responsive and nitric oxide (NO) modulated proteins Seedlings were subjected to cold

stress in a cold chamber An identical set is kept in the control conditions After stress

treatment protein extracts are prepared and NO mediated post-translational modifications (S-

nitrosylation S-glutathionylation and nitration) were analyzed To analyse the cold stress

specific changes the samples are subjected to 1-D and 2-DGE analysis Image analysis using

ImageMaster 2-D Platinum PDQuest detects cold stress induced up-regulated (U) down-

regulated (D) and noval (N) spots Using in-gel digestion differentially expressed proteins

were trypsin digested Gel free approach was also used for the identification Proteins were

identified using Mass spectrometry (MS) and validated for cold modulation SNOSID - SNO

site identification SNO-RAC- S-nitrosothiols using resin-assisted capture GRXs-

glutaredoxins GST- Glutathione S-transferase

Acc

epte

d A

rticl

e

Acc

epte

d A

rticl

eFigure 2 (A) Cold stress modulated functional categories in leaves roots anther and fruits

(B) Comparison of stress defense redox and signaling related cold responsive proteins

modulated by NO based posttranslational modifications

Acc

epte

d A

rticl

eFigure 3 An overview of the major metabolic pathways affected by cold stress and NO

Only cold responsive targets are enlisted in the table Enzymes which are enclosed in the

boxes are NO modulated either by S-nitrosylation nitration or glutathionylation

Figure 4 A hypothetical model showing NO signaling in cold stress As the plant perceives

cold stress membrane rigidification takes place Changes in cytoskeletal dynamics helps in

the activation of Ca2+

channels Increase in Ca2+

levels causes the activation of diacylglycerol

kinase (DGK) NO negatively regulates sphingolipids phoshorylation Enhanced NO

scavenges ROS by reacting with superoxide radicals (O2-) to produce peroxynitrite (ONOO

_)

and causes increased tyrosine nitration In addition NO also activates antioxidant enzyme

superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidase (APX) and glutathione

reductase (GR) Enhanced S-nitrosothiols (SNO) and S-nitrosoglutathione (GSNO)

contributes to increased S-nitrosylation of photosynthetic metabolic and stress defense and

Acc

epte

d A

rticl

eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

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eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

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e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

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eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

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e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

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eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

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e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

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e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

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e[102] Komatsu S Yamada E Furukawa K Cold stress changes the concanavalin A-

positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths

Amino Acids 2009 36 115ndash123

[103] Yang P F Li X J Liang Y Jing Y X et al Proteomic analysis of the response of

Liangyoupeijiu (Super High-Yield Hybrid Rice) seedlings to cold stress J Integr Plant Biol

2006 48 945minus951

[104] Rinalducci S Egidi M G Karimzadeh G Jazii F R Zolla L Proteomic analysis

of a spring wheat cultivar in response to prolonged cold stress Electrophoresis 2011 32

1807ndash1818

[105] Kosmala A Bocian A Rapacz M Jurczyk B Zwierzykowski Z Identification of

leaf proteins differentially accumulated during cold acclimation between Festuca pratensis

plants with distinct levels of frost tolerance J Exp Bot 2009 60 3595ndash3609

[106] Dumont E Bahrman N Goulas E Valot B et al A proteomic approach to

decipher chilling response from cold acclimation in pea (Pisum sativum L) Plant Sci 2011

180 86ndash98

[107] Gao F Zhou Y Zhu W Li X et al Proteomic analysis of cold stress-responsive

proteins in Thellungiella rosette leaves Planta 2009 230 1033ndash1046

[108] Jin Y Zhang C Yang H Yang Y et al Proteomic analysis of cold stress

responses in tobacco seedlings Afr J Biotechol 2011 10 18991ndash19004

[109] Evers D Legay S Lamoureux D Hausman J F et al Towards a synthetic view of

potato cold and salt stress response by transcriptomic and proteomic analyses Plant Mol

Biol 2012 78 503ndash514

[110] Long G Song J Deng Z Liu J Rao L Ptcorp gene induced by cold stress was

identified by proteomic analysis in leaves of Poncirus trifoliata (L) Raf Mol Biol Rep

2012 39 5859ndash5866

Acc

epte

d A

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e[111] Renaut J Lutts S Hoffmann L Hausman J F Responses of poplar to chilling

temperatures proteomics and physiological aspects Plant Biol 2004 6 81ndash90

[112] Koehler G Wilson R C Goodpaster J V Soslashnsteby A et al Proteomic study of

low-temperature responses in strawberry cultivars (Fragaria times ananassa) that differ in cold

tolerance Plant Physiol 2012 159 1787ndash1805

[113] Kjellsen T D Shiryaeva L Schroumlderw P Strimbeck R Proteomics of extreme

freezing tolerance in Siberian spruce (Picea obovata) J Proteomics 2010 73 965ndash975

[114] Chen J Tian L Xu H Tian D et al Cold-induced changes of protein and

phosphoprotein expression patterns from rice roots as revealed by multiplex proteomic

analysis Plant Omics J 2012 5 194-199

[115] Gai Y P Ji X L Lu W Han X J et al A novel late embryogenesis abundant like

protein associated with chilling stress in Nicotiana tabacum cv bright yellow-2 cell

suspension culture Mol Cell Proteomics 2011 10 M111010363

[116] Gammulla C G Pascovici D Atwell B J Haynes P A Differential metabolic

response of cultured rice (Oryza sativa) cells exposed to high- and low-temperature stress

Proteomics 2010 10 3001ndash3019

[117] Sanchez-Bel P Egea I Sanchez-Ballesta M T Sevillano L Proteome changes in

tomato fruits prior to visible symptoms of chilling injury are linked to defensive mechanisms

uncoupling of photosynthetic processes and protein degradation machinery Plant Cell

Physiol 2012 53 470ndash484

[118] Tanou G Job C Rajjou L Arc E et al Proteomics reveals the overlapping roles

of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity Plant J

2009 60 795ndash804

Acc

epte

d A

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e[119] Ortega-Galisteo A P Rodriacuteguez-Serrano M Pazmintildeo D M Gupta D K et al S-

Nitrosylated proteins in pea (Pisum sativum L) leaf peroxisomes changes under abiotic

stress J Exp Bot 2012 63 2089ndash2103

[120] Ito H Iwabuchi M Ogawa K The sugar-metabolic enzymes aldolase and triose-

phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana detection using

biotinylated glutathione Plant Cell Physiol 2003 44 655ndash660

[121] Zaffagnini M Michelet L Marchand C Sparla F et al The thioredoxin-

independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is

selectively regulated by glutathionylation FEBS J 2007 274 212ndash126

Figure Legends

Figure 1 Schematic diagram showing the methodology utilized for the identification of cold

stress responsive and nitric oxide (NO) modulated proteins Seedlings were subjected to cold

stress in a cold chamber An identical set is kept in the control conditions After stress

treatment protein extracts are prepared and NO mediated post-translational modifications (S-

nitrosylation S-glutathionylation and nitration) were analyzed To analyse the cold stress

specific changes the samples are subjected to 1-D and 2-DGE analysis Image analysis using

ImageMaster 2-D Platinum PDQuest detects cold stress induced up-regulated (U) down-

regulated (D) and noval (N) spots Using in-gel digestion differentially expressed proteins

were trypsin digested Gel free approach was also used for the identification Proteins were

identified using Mass spectrometry (MS) and validated for cold modulation SNOSID - SNO

site identification SNO-RAC- S-nitrosothiols using resin-assisted capture GRXs-

glutaredoxins GST- Glutathione S-transferase

Acc

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e

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eFigure 2 (A) Cold stress modulated functional categories in leaves roots anther and fruits

(B) Comparison of stress defense redox and signaling related cold responsive proteins

modulated by NO based posttranslational modifications

Acc

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eFigure 3 An overview of the major metabolic pathways affected by cold stress and NO

Only cold responsive targets are enlisted in the table Enzymes which are enclosed in the

boxes are NO modulated either by S-nitrosylation nitration or glutathionylation

Figure 4 A hypothetical model showing NO signaling in cold stress As the plant perceives

cold stress membrane rigidification takes place Changes in cytoskeletal dynamics helps in

the activation of Ca2+

channels Increase in Ca2+

levels causes the activation of diacylglycerol

kinase (DGK) NO negatively regulates sphingolipids phoshorylation Enhanced NO

scavenges ROS by reacting with superoxide radicals (O2-) to produce peroxynitrite (ONOO

_)

and causes increased tyrosine nitration In addition NO also activates antioxidant enzyme

superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidase (APX) and glutathione

reductase (GR) Enhanced S-nitrosothiols (SNO) and S-nitrosoglutathione (GSNO)

contributes to increased S-nitrosylation of photosynthetic metabolic and stress defense and

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eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

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eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

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e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

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e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

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eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

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e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

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e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

e[111] Renaut J Lutts S Hoffmann L Hausman J F Responses of poplar to chilling

temperatures proteomics and physiological aspects Plant Biol 2004 6 81ndash90

[112] Koehler G Wilson R C Goodpaster J V Soslashnsteby A et al Proteomic study of

low-temperature responses in strawberry cultivars (Fragaria times ananassa) that differ in cold

tolerance Plant Physiol 2012 159 1787ndash1805

[113] Kjellsen T D Shiryaeva L Schroumlderw P Strimbeck R Proteomics of extreme

freezing tolerance in Siberian spruce (Picea obovata) J Proteomics 2010 73 965ndash975

[114] Chen J Tian L Xu H Tian D et al Cold-induced changes of protein and

phosphoprotein expression patterns from rice roots as revealed by multiplex proteomic

analysis Plant Omics J 2012 5 194-199

[115] Gai Y P Ji X L Lu W Han X J et al A novel late embryogenesis abundant like

protein associated with chilling stress in Nicotiana tabacum cv bright yellow-2 cell

suspension culture Mol Cell Proteomics 2011 10 M111010363

[116] Gammulla C G Pascovici D Atwell B J Haynes P A Differential metabolic

response of cultured rice (Oryza sativa) cells exposed to high- and low-temperature stress

Proteomics 2010 10 3001ndash3019

[117] Sanchez-Bel P Egea I Sanchez-Ballesta M T Sevillano L Proteome changes in

tomato fruits prior to visible symptoms of chilling injury are linked to defensive mechanisms

uncoupling of photosynthetic processes and protein degradation machinery Plant Cell

Physiol 2012 53 470ndash484

[118] Tanou G Job C Rajjou L Arc E et al Proteomics reveals the overlapping roles

of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity Plant J

2009 60 795ndash804

Acc

epte

d A

rticl

e[119] Ortega-Galisteo A P Rodriacuteguez-Serrano M Pazmintildeo D M Gupta D K et al S-

Nitrosylated proteins in pea (Pisum sativum L) leaf peroxisomes changes under abiotic

stress J Exp Bot 2012 63 2089ndash2103

[120] Ito H Iwabuchi M Ogawa K The sugar-metabolic enzymes aldolase and triose-

phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana detection using

biotinylated glutathione Plant Cell Physiol 2003 44 655ndash660

[121] Zaffagnini M Michelet L Marchand C Sparla F et al The thioredoxin-

independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is

selectively regulated by glutathionylation FEBS J 2007 274 212ndash126

Figure Legends

Figure 1 Schematic diagram showing the methodology utilized for the identification of cold

stress responsive and nitric oxide (NO) modulated proteins Seedlings were subjected to cold

stress in a cold chamber An identical set is kept in the control conditions After stress

treatment protein extracts are prepared and NO mediated post-translational modifications (S-

nitrosylation S-glutathionylation and nitration) were analyzed To analyse the cold stress

specific changes the samples are subjected to 1-D and 2-DGE analysis Image analysis using

ImageMaster 2-D Platinum PDQuest detects cold stress induced up-regulated (U) down-

regulated (D) and noval (N) spots Using in-gel digestion differentially expressed proteins

were trypsin digested Gel free approach was also used for the identification Proteins were

identified using Mass spectrometry (MS) and validated for cold modulation SNOSID - SNO

site identification SNO-RAC- S-nitrosothiols using resin-assisted capture GRXs-

glutaredoxins GST- Glutathione S-transferase

Acc

epte

d A

rticl

e

Acc

epte

d A

rticl

eFigure 2 (A) Cold stress modulated functional categories in leaves roots anther and fruits

(B) Comparison of stress defense redox and signaling related cold responsive proteins

modulated by NO based posttranslational modifications

Acc

epte

d A

rticl

eFigure 3 An overview of the major metabolic pathways affected by cold stress and NO

Only cold responsive targets are enlisted in the table Enzymes which are enclosed in the

boxes are NO modulated either by S-nitrosylation nitration or glutathionylation

Figure 4 A hypothetical model showing NO signaling in cold stress As the plant perceives

cold stress membrane rigidification takes place Changes in cytoskeletal dynamics helps in

the activation of Ca2+

channels Increase in Ca2+

levels causes the activation of diacylglycerol

kinase (DGK) NO negatively regulates sphingolipids phoshorylation Enhanced NO

scavenges ROS by reacting with superoxide radicals (O2-) to produce peroxynitrite (ONOO

_)

and causes increased tyrosine nitration In addition NO also activates antioxidant enzyme

superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidase (APX) and glutathione

reductase (GR) Enhanced S-nitrosothiols (SNO) and S-nitrosoglutathione (GSNO)

contributes to increased S-nitrosylation of photosynthetic metabolic and stress defense and

Acc

epte

d A

rticl

eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

epte

d A

rticl

eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

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d A

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eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

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e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

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eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

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d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

e[119] Ortega-Galisteo A P Rodriacuteguez-Serrano M Pazmintildeo D M Gupta D K et al S-

Nitrosylated proteins in pea (Pisum sativum L) leaf peroxisomes changes under abiotic

stress J Exp Bot 2012 63 2089ndash2103

[120] Ito H Iwabuchi M Ogawa K The sugar-metabolic enzymes aldolase and triose-

phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana detection using

biotinylated glutathione Plant Cell Physiol 2003 44 655ndash660

[121] Zaffagnini M Michelet L Marchand C Sparla F et al The thioredoxin-

independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is

selectively regulated by glutathionylation FEBS J 2007 274 212ndash126

Figure Legends

Figure 1 Schematic diagram showing the methodology utilized for the identification of cold

stress responsive and nitric oxide (NO) modulated proteins Seedlings were subjected to cold

stress in a cold chamber An identical set is kept in the control conditions After stress

treatment protein extracts are prepared and NO mediated post-translational modifications (S-

nitrosylation S-glutathionylation and nitration) were analyzed To analyse the cold stress

specific changes the samples are subjected to 1-D and 2-DGE analysis Image analysis using

ImageMaster 2-D Platinum PDQuest detects cold stress induced up-regulated (U) down-

regulated (D) and noval (N) spots Using in-gel digestion differentially expressed proteins

were trypsin digested Gel free approach was also used for the identification Proteins were

identified using Mass spectrometry (MS) and validated for cold modulation SNOSID - SNO

site identification SNO-RAC- S-nitrosothiols using resin-assisted capture GRXs-

glutaredoxins GST- Glutathione S-transferase

Acc

epte

d A

rticl

e

Acc

epte

d A

rticl

eFigure 2 (A) Cold stress modulated functional categories in leaves roots anther and fruits

(B) Comparison of stress defense redox and signaling related cold responsive proteins

modulated by NO based posttranslational modifications

Acc

epte

d A

rticl

eFigure 3 An overview of the major metabolic pathways affected by cold stress and NO

Only cold responsive targets are enlisted in the table Enzymes which are enclosed in the

boxes are NO modulated either by S-nitrosylation nitration or glutathionylation

Figure 4 A hypothetical model showing NO signaling in cold stress As the plant perceives

cold stress membrane rigidification takes place Changes in cytoskeletal dynamics helps in

the activation of Ca2+

channels Increase in Ca2+

levels causes the activation of diacylglycerol

kinase (DGK) NO negatively regulates sphingolipids phoshorylation Enhanced NO

scavenges ROS by reacting with superoxide radicals (O2-) to produce peroxynitrite (ONOO

_)

and causes increased tyrosine nitration In addition NO also activates antioxidant enzyme

superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidase (APX) and glutathione

reductase (GR) Enhanced S-nitrosothiols (SNO) and S-nitrosoglutathione (GSNO)

contributes to increased S-nitrosylation of photosynthetic metabolic and stress defense and

Acc

epte

d A

rticl

eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

epte

d A

rticl

eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

e

Acc

epte

d A

rticl

eFigure 2 (A) Cold stress modulated functional categories in leaves roots anther and fruits

(B) Comparison of stress defense redox and signaling related cold responsive proteins

modulated by NO based posttranslational modifications

Acc

epte

d A

rticl

eFigure 3 An overview of the major metabolic pathways affected by cold stress and NO

Only cold responsive targets are enlisted in the table Enzymes which are enclosed in the

boxes are NO modulated either by S-nitrosylation nitration or glutathionylation

Figure 4 A hypothetical model showing NO signaling in cold stress As the plant perceives

cold stress membrane rigidification takes place Changes in cytoskeletal dynamics helps in

the activation of Ca2+

channels Increase in Ca2+

levels causes the activation of diacylglycerol

kinase (DGK) NO negatively regulates sphingolipids phoshorylation Enhanced NO

scavenges ROS by reacting with superoxide radicals (O2-) to produce peroxynitrite (ONOO

_)

and causes increased tyrosine nitration In addition NO also activates antioxidant enzyme

superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidase (APX) and glutathione

reductase (GR) Enhanced S-nitrosothiols (SNO) and S-nitrosoglutathione (GSNO)

contributes to increased S-nitrosylation of photosynthetic metabolic and stress defense and

Acc

epte

d A

rticl

eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

epte

d A

rticl

eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

eFigure 2 (A) Cold stress modulated functional categories in leaves roots anther and fruits

(B) Comparison of stress defense redox and signaling related cold responsive proteins

modulated by NO based posttranslational modifications

Acc

epte

d A

rticl

eFigure 3 An overview of the major metabolic pathways affected by cold stress and NO

Only cold responsive targets are enlisted in the table Enzymes which are enclosed in the

boxes are NO modulated either by S-nitrosylation nitration or glutathionylation

Figure 4 A hypothetical model showing NO signaling in cold stress As the plant perceives

cold stress membrane rigidification takes place Changes in cytoskeletal dynamics helps in

the activation of Ca2+

channels Increase in Ca2+

levels causes the activation of diacylglycerol

kinase (DGK) NO negatively regulates sphingolipids phoshorylation Enhanced NO

scavenges ROS by reacting with superoxide radicals (O2-) to produce peroxynitrite (ONOO

_)

and causes increased tyrosine nitration In addition NO also activates antioxidant enzyme

superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidase (APX) and glutathione

reductase (GR) Enhanced S-nitrosothiols (SNO) and S-nitrosoglutathione (GSNO)

contributes to increased S-nitrosylation of photosynthetic metabolic and stress defense and

Acc

epte

d A

rticl

eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

epte

d A

rticl

eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

eFigure 3 An overview of the major metabolic pathways affected by cold stress and NO

Only cold responsive targets are enlisted in the table Enzymes which are enclosed in the

boxes are NO modulated either by S-nitrosylation nitration or glutathionylation

Figure 4 A hypothetical model showing NO signaling in cold stress As the plant perceives

cold stress membrane rigidification takes place Changes in cytoskeletal dynamics helps in

the activation of Ca2+

channels Increase in Ca2+

levels causes the activation of diacylglycerol

kinase (DGK) NO negatively regulates sphingolipids phoshorylation Enhanced NO

scavenges ROS by reacting with superoxide radicals (O2-) to produce peroxynitrite (ONOO

_)

and causes increased tyrosine nitration In addition NO also activates antioxidant enzyme

superoxide dismutase (SOD) catalase (CAT) ascorbate peroxidase (APX) and glutathione

reductase (GR) Enhanced S-nitrosothiols (SNO) and S-nitrosoglutathione (GSNO)

contributes to increased S-nitrosylation of photosynthetic metabolic and stress defense and

Acc

epte

d A

rticl

eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

epte

d A

rticl

eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

eredox related proteins NO positively regulates CBF dependent COR gene signaling by

activating CBF1 and CBF3 Unaffected ZAT12 by NO is shown with (harr) The regulation of

ICE1 and HOS1 by NO is not known and shown with () ABA dependent cold signaling is

positively regulated by the activation of bZIP factors by NO NO also participates in

increasing proline sugars dehydrins and lipocalins accumulation which helps in membrane

stabilization and cold acclimation

Acc

epte

d A

rticl

eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

eTable 1 A summary of proteomics studies performed to investigate the effect of coldfreezing stress on plants

S No Organism Tissue (age) Techniques Temperature amp stress duration Reference

1 Oryza sativa Leaves (2 weeks) 2-DE-MS 15 degC 10 degC amp 5 degC for 24 hrs [64]

2 Oryza sativa Leaves (3 weeks old) 2-DE-MS 6degC for 6 hrs amp 24 hrs [63]

3 Oryza sativa Leaves (4-5 leaf stage) Labell free and iTRAQ daynight 14degC12degC for 48 72 and 96 hrs [49]

4 Oryza sativa Leaves (3weeks old) 2-DE-MS 5degC for 12 24 36 h and 10degC for 24 and 72 h at 60 relative

humidity

[101]

5 Oryza sativa Seedlings (Leaf sheath

leaf blade and root 2

weeks old)

2-DE-MS 5 degC for 48 h under glimmer light [52]

6 Oryza sativa Basal part of leaf sheaths

(2 weeks old)

2-DE-MS glycosylation 5 degC for 48 h under glimmer light [102]

7 Liangyoupeijiu

(Super High-Yield

Hybrid Rice)

Leaves (14 days) 2-DE-MS 4degC for 6 hrs [103]

8 Triticum aestivum L

em

Thell

Leaves (14 days) 2-DE-MS 2 degC at 200 micromolm2s 12 hrs photoperiod [68]

9 Spring wheat (Triticum

aestivum cv kohdast)

Leaves (14 days) 2-DE-MS 4degC for 42 days at 12 hrs photoperiod 300 micromolm2s [104]

10 Triticum aestivum

(winter wheat)

Crown (3 leaf stage) 2-D DIGE-MS 6degC for 3 21 amp 84 days at 12 hrs photoperiod 400 micromolm2s [78]

11 Triticum aestivum Crown (5 weeks) 2-DE-MS 3degC for 3 weeks at 12 h photoperiod at 310 micromolm2s and then

shifted to -3degC in the dark for 6 h 1 d and 3 d

[74]

12 Arabidopsis Leaves (5 weeks) 2-D DIGE-MS 6 degC amp10 degC for 1 week [66]

13 Festuca pratensis Leaves 2-DE-MS 2 8 amp 26 hrs and 3 5 7 14 amp 21 d at 4degC 2degC 1014 h

photoperiod and 200 micromolm2s

[105]

14 Pisum sativum Leaves stems and roots 2-DE-MS [106]

15 Lathyrus sativus L Leaves (5 weeks) 2-DE-MS 4degC for 6 12 1824 30 and 36 h [72]

16 Helianthus annuus Leaves (6 weeks) Labell free shotgun

proteomics (spectral

counting)

Temperature was decreased progressively during a week from

2515degC daynight to 155degC daynight at 12 h photoperiod 200

micromolm2s

[67]

17 Thellungiella halophila Leaves (4 weeks) 2-DE-MS 54degC daynight for 6 hrs 2 5 amp 24 days [107]

18 Nicotiana tabacum Leaves (5 weeks) 2-DE-MS 4degC for 4 hrs [108]

19 Solanum tuberosum Leaves (2 months) 2-D DIGE-MS 72degC daynight for 3 and 8 days [109]

20 Poncirus trifoliata Leaves (2 years) 2-DE-MS -6 degC for 50 and 80 min [110]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

e21 Poplar Leaves (3 months) 2-DE-MS 4degC for 7 amp 14 days at 16 hrs photoperiod 150 micromolm2s [111]

22 Strawberry

(Fragariatimesananassa)

Crown (6 weeks) 2-DE-MS 2degC for 6 weeks at 1014 h daynight at 90 micromolm2s [112]

23 Picea obovata Needles 2-D DIGE-MS [113]

24 Physcomitrella patens Leafy gametophores (3

weeks)

2-DE-MS 0 degC for 12 amp 3 days at 16 h 8 h 55 micromolm2s [85]

25 Oryza sativa Roots (2 weeks) 2-DE-MS 10degC for 24 amp 72 hrs at 60 humidity [51]

26 Oryza sativa Roots (2 weeks) Phosphoproteome analysis

2-DE-MS

15 and 6 degC for 12 h [114]

27 Chicory Roots 2-DE-MS Between 5 degC and freezing [53]

28 Nicotiana tabacum cv

Bright Yellow-2

Cell

Suspension Culture

2-DE-MS 4degC for 12 hrs [115]

29 Oryza sativa Cultured cells (2 months) Shotgum proteomics 12 degC amp 20 degC for 3 days [116]

30 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[55]

31 Oryza sativa Anther 2-DE-MS 12degC 70 relative humidity for 4 days at 12 hrs photoperiod 330

micromolm2s

[56]

32 Zea mays Anthers 2-DE-MS 7degC for 12 days [54]

33 Solanum lycopersicum Fruits 2-D DIGE-MS 2degC for 17 amp 20 days followed by 20degC for 48 hrs [117]

34 Peach Fruit mesocarp 2-D DIGE-MS [71]

35 Hirado Buntan

Pummelo (Citrus

grandis times C paradis)

Fruit (Juice sacs) 2-DE-MS 8ndash10 degC for 24 48 72 96 and 120 days after harvest at relative

humidity of 85ndash90

[73]

36 Glycine max Seeds 2-DE-MS 4degC for 24 hrs in dark [57]

37 Prunus persica Bark (1 year) 2-D DIGE-MS 5degC for 3 amp 5 weeks at with 8 h light16 h dark cycles [84]

38 Cotton Fibres 2-DE-MS 10 15 and 20 days post-anthesis [58]

Table 2 A summary of proteomics studies performed in sub-cellular organelles to investigate the effect of cold stress

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

eTable 2

Functional

classification

Cold stress responsive proteins NO based PTMs Cold

Modulation

Tissue in which cold modulation

was analyzed

Stress Glyoxalase I S-nitrosylation in cold stress (unpublished) Nitration

in salt stress [50]

[51 72 73] Rice roots grass pea leaves citrus

fruits

Stress Cold-regulated protein (cor15a cor 18) S-nitrosylation by NO donor [83] [73 104] Wheat seedlings citrus fruits

Stress Ethylene-responsive GTP-binding

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Stress 70 kDa heat shock protein S-nitrosylation by NO donor [83 50] in vivo S-

glutathionylation [51 43] in vivo nitration [24]

[51 55 64

72 73 85

104 111

116]

Poplar leaves rice anthers leaves

cell culture roots moss leafy

gametophore wheat seedlings

citrus fruits

Stress Small Heat shock protein S-nitrosylated in salt stress [50] [111] Poplar leaves

Stress Putative calreticulin precursor in vivo S-nitrosylation [77] in vivo S-glutathionylation

[42]

[51]

Rice roots

Defense Protease inhibitor S-nitrosylation in salt stress [118] and by NO donor

[75]

[106] Pea leaves root and stem

Defense Putative major latex protein S-nitrosylation in cold stress [7] and by NO donor [83] [66] Arabidopsis leaves

Defense Cysteine protease inhibitor S-nitrosylation in vivo [77] and in salt stress [118] [63 73 107] Rice seedlings Thellungiella

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

e halophila rosette leaves citrus fruits

Defense Chitinase S-nitrosylation in vivo [77] and by NO donor [75 76]

Nitration in salt stress [50]

[113] Citrus fruits

Defense Disease resistance

response protein

S-nitrosylation by pathogen infection [76] in vivo

nitration [24]

[42 74] Soyabean seeds pea leaves root

and stem

Defense Polygalacturonase inhibitor-like

protein

S-nitrosylation by pathogen infection [76] [67] Sunflower leaves

Defense Glutathione S-transferase II S-nitrosylation in salt stress [118] S-gutathionylation

[43] Nitration in salt stress [50]

[39 42 64

80 81]

Rice leaves peach fruit mesocarp

soyabean seeds rice cell culture

grass pea leaves

Defense Glutathione S-transferase I Nitration in salt stress [50] [67] Sunflower leaves

Defense RNA-binding domain in vivo S-glutathionylation [42] [67 104

107]

Thellungiella halophila rosette

leaves wheat seedlings sunflower

leaves

Defense Similarity to RNA binding protein S-nitrosylation by NO donor [83] [67] Sunflower leaves

Defense Serine protease S-nitrosylated in salt stress [50] [67] Sunflower leaves

Defense Putative chaperonin 60 precursor S-nitrosylation by NO donor [83] [72] Rice seedlings

Defense Putative 10kd chaperonin S-nitrosylation by NO donor [83] S-glutathionylation

[89]

[67] Sunflower leaves

Defense Pathogenesis-related protein PR- 10a S-nitrosylation by NO donor [75] [81] Grass pea leaves

Defense Putative glycine-rich RNA binding S-nitrosylated by NO donor [75] in vivo nitration [24] [67] Sunflower leaves

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

eprotein

Signaling Calcium-binding protein calmodulin-

like protein

in vivo S-nitrosylation [77] tyrosine nitrated in heat

stress [46]

[15] Rice leaves

Signaling Peptidyl-prolyl cis-trans isomerase S-nitrosylated in vivo [77] and in cold stress

(unpublished) Nitration in salt stress [50]

[108] Tobacco leaves

Signaling GTP-binding nuclear protein Ran-1

(Ras-related nuclear protein 1)

S-nitrosylation in salt stress [50] [116 67] Rice cell culture sunflower leaves

Signaling 14-3-3 protein Tyrosine nitrated in heat stress [46] [81] Grass pea leaves

Redox Thioredoxin H F and M type S-nitrosylation in vivo [77] and by pathogen infection

[75 76] in vitro S-glutathionylation [89]

[63 67 71

81 98]

Rice seedlings meadow fescue

leaves peach fruit mesocarp grass

pea leaves Sunflower leaves

Redox L-ascorbate peroxidase S-nitrosylation by NO donor [75 76] [4053 55

63 67 71

74 73 104

111 116]

Poplar leaves rice anthers and

seedlings chicory roots peach fruit

mesocarp siberian spruce needles

rice cell culture pea leaves root and

stem wheat seedlings sunflower

leave maize anthers

Redox 2-cys peroxiredoxin chloroplast S-nitrosylation by pathogen infection [75 76] in vivo

and in vitro S-glutathionylation [42 43]

[63 107] Rice seedlings Thellungiella

halophila rosette leaves

Redox Peroxiredoxin-2E chloroplast

precursor

S-nitrosylation by NO donor [83] [107] Thellungiella halophila rosette leaves

Redox Peroxiredoxin S-nitrosylation in vivo [77] and by NO donor [83] [67] Sunflower leaves

Redox Dehydroascorbate reductase S-nitrosylation by NO donor [75] in vitro S- [104] Wheat seedlings

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

e glutathionylation [43] nitration in salt stress [50]

Redox Monodehydroascorbate reductase S-nitrosylation in vivo [77] and in salt stress [50] in

vitro S-glutathionylation [43]

[67] Sunflower leaves

Redox Catalase S-nitrosylation by pathogen infection [76] and by NO

donor [75 119] in vivo nitration [24]

[53 71113] Chicory roots peach fruit mesocarp

siberian spruce needles

Redox Basic peroxidase precursor 1 S-nitrosylation by NO donor [76] [67] Sunflower leaves

Redox Annexin S-nitrosylation by NO donor [75 76] [71 111] Poplar leaves peach fruit mesocarp

Redox Superoxide dismutase S-nitrosylation in vivo [77] in salt stress [50] by NO

donor [75] tyrosine nitrated in salt stress [50]

[53 71] Chicory roots peach fruit mesocarp

Redox Glutaredoxin S-nitrosylation in vivo [77] and by NO donor [75] [116] Rice cell culture

Redox Protein disulfide isomerase S-nitrosylation in salt stress [118] by pathogen

infection [76]

[73] Citrus fruits

Table 3 Compilation of cold responsive proteins identified to be S-nitrosylated Tyrosine nitrated and S-glutathionylated

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]

Acc

epte

d A

rticl

e

Sno Plant Tissue (age) Organelle Technique Treatment

tempamp

duration

References

1 Arabidopsis thaliana Leaves (3 weeks) Nucleus 2-DE-MS 4 degC 36 hrs [93]

2 Arabidopsis thaliana Leaves (20 days) Plasma

membrane

2-DE-MS 4 degC 6 hrs under

dark

[61]

3 Oryza sativa Roots (2 weeks) Plasma

membrane

2-DE-MS 5 degC 48 hrs [70]

4 Arabidopsis thaliana Leaves (30 days) Chloroplast 2-D DIGE-MS 5 degC for 1 10 amp 40

days

[62]

5 Pisum saivum Leaves (10 days) Mitochondria 2-DE-MS 2 degC 8-h

photoperiod at

125microEm2 S

[90]

6 Hippophae

rhamnoides

Shoot (20 days) Apoplast 2-DE-MS 4 degC for 1 amp 5

days

[69]