27Polyamines in Developing Stress-Resistant CropsFrancisco Marco, Rubén Alc�azar, Teresa Altabella, Pedro Carrasco, Sarvajeet Singh Gill,Narendra Tuteja, and Antonio F. Tiburcio

Polyamines (PAs) are small protonated compounds with key roles in plant devel-opment and physiological processes. PAs may also function as stress messengers inplant responses to different stress signals. Recent studies using exogenous appli-cation of polyamines and more contemporary genetic manipulation of polyaminelevels in crops andmodel species point to their involvement in stress protection. Thedifferent mechanisms by which polyamines exert their functions are presently beingunraveled and involve different modes of action that are summarized in this chapter.Polyamines are integrated with other stress-related hormone pathways, such asabscisic acid (ABA), reactive oxygen species (ROS) signaling, nitric oxide, andregulation of ion channels that are now being elucidated. Also, polyamines areimplicated in the transcriptional regulation to abiotic and biotic stresses as revealedin recent global transcriptome analyses. The genetic manipulation of polyaminelevels has been proven to be an efficient tool for enhancing stress tolerance in manyplant species. A number of examples and their potential application to crops for asustainable agriculture are discussed in this chapter, along with the most recentadvances in our understanding of the regulation and mode of action of polyamines.

27.1Introduction

27.1.1PA Biosynthesis and Catabolism in Plants

Plants live in an ever-changing and often unpredictable environment that repre-sents the major limiting factors for agricultural crop productivity. Plants, unlikeanimals, cannot move and therefore encounter a variety of environmental stressesthroughout their life cycle. It is predicted that the environmental stresses willbecome more intense and frequent with climate change, especially global warming.Among abiotic stresses, cold, heat, salinity, and drought adversely affect plantgrowth and productivity and restrict the crops to reach their full genetic potential [1].

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

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World population is also increasing at an alarming rate and expected to reacharound 10 billion by 2050, which will witness serious food shortages. Therefore,reducing crop loss is a major challenge to meet the increasing food demand [2].Plants employ various strategies to cope with the ever-changing environmentalfluctuations [3].

Polyamines (PAs) are a group of polycationic amine-containing compoundswhosemost predominant forms are the diamine putrescine (Put), triamine spermidine(Spd), and tetramine spermine (Spm) that play a pivotal role in the regulation ofdevelopmental and physiological processes in plants [4]. Put, Spd, and Spm mole-cules differ in the number of aminopropyl moieties added to the carbon skeleton ofPut, and thus they differ in their number of positively charged amine groups at thephysiological pH of the cell. Metabolic studies indicate that the intracellular levels ofPAs in plants are mostly regulated by anabolic and catabolic processes (Figure 27.1),as well as by their conjugation to hydroxycinnamic acids and other macromoleculessuch as proteins and DNA.

The PA biosynthetic pathway starts with the synthesis of the diamine Put. Inmammals and fungi, Put biosynthesis is exclusively derived from ornitine (Orn)decarboxylation in a reaction catalyzed by ornithine decarboxylase (ODC). Plants andbacteria can also synthesize Put via an alternative pathway involving arginine (Arg)decarboxylation by arginine decarboxylase (ADC), aswell as two additional successive

Figure 27.1 Polyamine biosynthesis andcatabolism in plants. Biosynthetic pathways areindicated in black and degradation routes ingrey. ACCs, ACC synthase; ACCox, ACC oxidase;ADC, arginine decarboxylase; AIH, agmatineiminohydrolase; CPA, N-carbamoylputrescineamidohydrolase; DAO, diamine oxidase; 1,3-DAP, 1,3-diaminopropane; dcSAM,

decarboxylated S-adenosylmethionine; ODC,ornithine decarboxylase; PAO, polyamineoxidase; SAM, S-adenosylmethionine; SAMs,S-adenosylmethionine synthase; SAMDC,S-adenosylmethionine decarboxylase; SPDS,spermidine synthase; SPMS, sperminesynthase.

624j 27 Polyamines in Developing Stress-Resistant Crops

steps involving agmatine iminohydrolase (AIH) and N-carbamoylputrescine ami-dohydrolase (CPA) activities. Put serves as a precursor of Spd andSpmby consecutiveadditions of aminopropyl groups catalyzed, respectively, by Spd synthase (SPDS) andSpm synthase (SPMS). Both enzymes use decarboxylated S-adenosylmethionine(dcSAM) as donor of aminopropyl moieties, which is formed by decarboxylation ofSAM in a reaction catalyzed by SAM decarboxylase (SAMDC) (Figure 27.1).

PAs are degraded by oxidative deamination in reactions catalyzed by amineoxidases, in particular diamine oxidases (DAOs) and PA oxidases (PAOs). DAOsdisplay high affinity for diamines, similar to Put, producingD1-pyrroline, H2O2, andammonia (Figure 27.1).D1-pyrroline is catabolized into c-aminobutyric acid (GABA)(Figure 27.1), which is ultimately converted into succinic acid, a component of theKrebs cycle. PAOs oxidize secondary amine groups fromSpd and Spm, leading to theformation of 4-aminobutanal or (3-aminopropyl)-4-aminobutanal, along with 1,3-diaminopropane (DAP) andH2O2 (Figure 27.1). Spm could also be backconverted toSpd by PAOs with concomitant production of 3-aminopropanal and H2O2. There-fore, the PA metabolic pathway is also interconnected with other metabolic routesinvolved in the formation of various signaling molecules and metabolites that arerelevant to plant stress responses such as ethylene, GABA, or H2O2 (Figure 27.1) [5].

Many evidences point to the requirement of Put and Spd for plant life anddevelopment. Depletion of Put and Spd levels by genetic or chemical means islethal in yeast, protists, and plants [6–9]. Indeed, all living organisms analyzed so farcontain endogenous pools of Put and Spd. Conversely, Spm-deficient organismsseem viable but show different degrees of dysfunction, thus suggesting an importantinvolvement of Spm in growth and developmental processes [10–13].

In plants, PAs have been implicated in a wide array of fundamental processes suchas cell cycle, transcriptome regulation, hormone signaling, plant growth and devel-opment, and response to biotic and abiotic stresses [14–21].

27.2PAs and Stress

The first observation on the effects of stress in PA levels in plants was reported byRichards and Coleman [22], who showed an increase in the endogenous levels ofPut in oat plants grown under potassium starvation. Since then, a large number ofstudies have shown an increase in PA levels in response to different biotic andabiotic stresses [14, 16, 23]. Stress-triggered PA accumulation correlateswith enhanced tolerance to different stresses, such as salinity [24–27], chilling[27, 28], osmotic and acidic stresses [29], radiation-induced oxidative stress [30], andso on [23].

Also, early studies based on the application of exogenous PAs or PA biosynthesisinhibitors have also been useful to identify correlation between PA stress accumu-lation and plant tolerance [16]. Nevertheless, it has to be noted that exogenousapplication of PAs may have certain limitations, such as differences in the uptakerates between replicates, the possible deleterious effects of PAs when applied to

27.2 PAs and Stress j625

membranes at high doses, and an insufficient specificity of the inhibitor applied insome instances, determined frequently by differences in the localization of theinhibitor and the target enzyme [31].

Since the identification of the oat ADC by Bell and Malmberg [32], many genescoding for enzymes involved in PAmetabolism have been cloned from several plantspecies and their expression under stress conditions analyzed [14, 33]. Reports fromthose experiments show that some of the PA biosynthetic genes raise theirexpression levels in response to stress, although with different kinetics. Some PAbiosynthetic genes are rapidly induced shortly after stress treatment and undergo acontinuous rise or a minor change with a prolonged period of stress. Conversely,others are induced only when the stress is exerted for a certain period. Theseobservations indicate a differential regulation of PA biosynthetic genes duringstress, consistent with different pathways involved in the regulation of PA biosyn-thesis under stress [34]. When trying to combine transcriptional profiles fromindependent experimental designs, the different kinetics may depend on severalfactors, such as plant species, duration, and intensity of stress or stress sensitivity ofthe experimental materials [24, 25, 35, 36]. Hence, rather than analyzing singlegenes in the PA pathway, a broader approach that aims to analyze the whole PAbiosynthetic transcriptome is more informative for the study of gene kinetics.Unfortunately, there are very few examples where these approaches have beenundertaken [34, 37–40].

27.3Transgenic Modifications of PA Biosynthetic Route and Improvementof Stress Tolerance

The identification and cloning of the genes coding for PA biosynthetic enzymes hasalso allowed the generation of transgenic plantswith altered endogenousPA levels, inorder to overcome the problems arising from the use of exogenous PAs or the lack ofspecificity of certain PA biosynthetic inhibitors. Table 27.1 summarizes a number ofexamples of overexpression ofODC,ADC, SAMDC, and SPDS in rice, tobacco, pear,sweet potato, andArabidopsis during over the years, with different results concerningthe modification of one or more specific PAs, but all showing in common anenhanced tolerance against a broad spectrum of stress conditions (Table 27.1).Enhanced tolerance always correlated with elevated levels of Put and/or Spd andSpm. Transgenic rice plants carrying oat ADC under control of an abscisic acid(ABA)-inducible promoter showed higher ADC activity, higher Put level, andincreased biomass under salt stress than wild-type plants [41]. Similarly, constitutiveoverexpresssion ofDatura stramonium ADC gene in rice produced transgenic plantsthat accumulated higher levels of Spd and Spm than the wild type when exposed todrought stress. These lines also showed an improvement in drought tolerance, with alower degree of chlorophyll loss and leaf curling than thewild type [29].More recently,constitutive homologous overexpression of ADC genes in Arabidopsis also leads totransgenic plants with elevated Put levels and resistant to freezing conditions [42] and

626j 27 Polyamines in Developing Stress-Resistant Crops

Table27.1

Abioticstress

tolerancein

plan

tswith

overprod

uctio

nof

PAsob

tained

bytran

sgen

icmod

ificatio

n.

Gene

Source

Tran

sgenic

plan

tOverexpression

Overprodu

ction

Tolerance

Reference

ADC

Oat

Rice

Indu

cible

Put

Salt

[41]

D.stram

onium

Rice

Con

stitutive

Spdan

dSp

mDrough

t[29]

Arabidopsis(ADC1)

Arabidopsis

Con

stitutive

Put

Freezing

[42]

Arabidopsis(ADC2)

Arabidopsis

Con

stitutive

Put

Drough

t[43]

ODC

Mou

seTobacco

Con

stitutive

Put

Salt

[44]

SAMDC

Tritordeum

Rice

Indu

cible

Spdan

dSp

mSalt

[26]

Human

Tobacco

Con

stitutive

Putan

dSp

dSalt,

osmotic

[45]

Carnation

Tobacco

Con

stitutive

Put,Sp

d,an

dSp

mBroad

spectrum

[46]

Yeast

Tomato

Con

stitutive

Spdan

dSp

mHeat

[47]

Arabidopsis(SAMDC1)

Arabidopsis

Con

stitutive

Spm

Salt

[67]

SPDS

C.fi

cifolia

Arabidopsis

Con

stitutive

Spd

Broad

spectrum

[48]

C.fi

cifolia

Sweetpo

tato

Con

stitutive

Spd

Broad

spectrum

[49]

App

lePear

Con

stitutive

Spd

Heavy

metal

(Al)

[50]

ACCs

Carnation

Tobacco

Antisense

Putan

dSp

dBroad

spectrum

[51]

ACCox

Carnation

Tobacco

Antisense

Putan

dSp

dBroad

spectrum

[51]

Source:A

dapted

from

Refs[4,5

].

27.3 Transgenic Modifications of PA Biosynthetic Route and Improvement of Stress Tolerance j627

drought stress [43]. Salt tolerance has also been obtained in tobacco plants over-expressing mouse ODC, which showed higher Put levels, a better germinationfrequencies, and a less degree of wilting in salt than wild-type plants [44].

SAMDC cDNAshave been also used to generate transgenic plantswith elevatedPAlevels (Table 27.1). Constitutive overexpression of Tritordeum SAMDC gene in riceresulted in a three- to fourfold increase in Spd and Spm levels in the transformedplants [26]. Stress tolerance of those plants was demonstrated by their normal growthand development under NaCl stress. The tobacco plants obtained by Waie andRajam [45] are another example of heterologous constitutive SAMDC overexpressionwhere a human SAMDC gene was driven by a constitutive CaMV35S promoter. Thetransgenic tobacco lines obtained showed higher Spd and Put levels, as well astolerance to salt and drought stresses [45]. More recently, transgenic tobacco plantsoverexpressing carnation SAMDC also showed a broad-spectrum tolerance to abioticstresses [46]. Transgenic tomato plants with high levels of Spm and Spd and tolerantto heat stress have been obtained by constitutive overexpression of yeast SAMDCgene [47]. Also, homologous overexpression of SAMDC1 gene inArabidopsis leads toelevated Spm levels and enhanced tolerance to salt stress [67].

Transgenic plants overexpressing SPDS share common features of elevated Spdlevels aswell as broad-spectrum stress tolerance (Table 27.1). Thus, overexpression ofSPDS fromCucurbita ficifolia in Arabidopsis enhanced tolerance to chilling, freezing,drought, salinity, osmosis, and paraquat [48]. Transformation of the same gene intosweet potato (Ipomoea batatas) produced transgenic plants withmore tolerance to saltand drought than the wild type [49]. Higher Spd titers are also found in transgenicpear plants overexpressing apple MDSPDS1 gene, being also more tolerant thanwild-type plants when exposed to AlCl3 long-term stress [50].

On the other hand,Wi and Park [51] employed an alternative way to raise PA levelsby favoring the flux of SAM to PAs using antisense silencing of ethylene biosynthesisgenes ACC synthase and ACC oxidase in tobacco. Transgenic plants obtained raisedPut and Spd levels and increased their tolerance to oxidative, high salinity, and acidstresses [51]. Previously, high PA levels were also found in a tobacco DFMO-resistantline, which was also resistant to acidic stress conditions [52].

In summary, all these examples show the correlation between accumulation of Putand Spd and Spmwith stress tolerance, often with a broad spectrum of stresses. Thiscorrelation is also reinforced by the results obtained from loss-of-functionmutationsin PA biosynthetic genes. For example, EMS mutants of Arabidopsis thaliana spe1-1and spe2-1 (which map to ADC2) showing reduced ADC activity are deficient in PAaccumulation after acclimation to high NaCl concentrations and exhibit moresensitivity to salt stress [53]. Another study shows that a Ds insertion mutant(adc2-1), with Put levels diminished up to 75% of wild-type plants, is more sensitiveto salt stress, whereas its salt-induced injury is partly reverted by the addition ofexogenous Put [54]. Other ADC1 (adc1-2, adc1-3) and ADC2 (adc2-3, adc2-4) mutantalleles are more sensitive to freezing, and this phenotype is partially rescued byadding exogenous Put [37]. On the other hand, acl5/spms Arabidopsis doublemutantsthat do not produce Spm and thermospermine are hypersensitive to salt and droughtstresses, and the phenotype is mitigated by application of exogenous Spm [55].

628j 27 Polyamines in Developing Stress-Resistant Crops

27.4Possible Mechanisms of PA Action in Stress Responses

Taken together, results presented in the above sections indicate that elevatedPA levelsrepresent a stress-induced protective response with a protective role. However, theprecise mechanism of action by which PAs could protect plants from challengingenvironmental conditions remains unclear, although some progress has beenmade [4, 56].

Classically, the most common explanation for protective roles of PAs have beenrelated to their chemical structure: the polycationic nature of PAs at physiological pHenables them tomodulate ion balance of the cell and interact with anionicmolecules,such asDNA,RNA, proteins, andmembrane lipids [57, 58]. PAbinding to proteins ornucleic acids could not only protect them from degradation but also provide amoleculewith themost stable conformation under stress conditions. A large numberof evidences suggested that exogenous application of PAs (di-, tri-, and tetraamines)were shown to stabilize plant cell membranes, protecting them from damage understress conditions [33, 59], and endogenous PAs are also suggested to participate insustaining membrane integrity [60].

Also, an antioxidative role has been proposed for PAs due to a combination of theirpossible role as radical-scavengingmolecules bymeans of their dual anion and cationbinding properties [61] and their capability to inhibit both lipid peroxidation [62] andmetal-catalyzed oxidative reactions [63]. Spm, Spd, and Put all reduce level ofsuperoxide radicals generated by senescing plant cells [64]. Alternatively, PA catab-olism produces H2O2, a signaling molecule that can enter the stress signal trans-duction chain promoting an activation of an antioxidative defense response.Owing tothe fact that this peroxide production could also be a source of oxidant species, the roleof PAs acting as antioxidants is still a matter of debate [23].

On the other hand, more recent data show that modification of endogenous PAlevels alters the expression of an important number of genes, most of them stressrelated. Some of these stress-related genes could promote the synthesis of moreprotective compounds and render stress tolerance. This is seen, for example, intobacco SAMDC overexpressing plants that have higher levels of expression ofseveral antioxidant enzymes, such as ascorbate peroxidase, superoxide dismutase,and glutathione S-transferase [46]. Microarray analysis of Arabidopsis plants over-expressing SPDS shows that those transgenic plants have higher expression levels ofsome stress-related transcription factors, such as DREB, WRKY, B-box zinc fingerproteins, NAM proteins, and MYB, along with stress-regulated genes, such as low-temperature-induced protein 78 (LTI78 or rd29A) [48]. More recently, analysis oftranscriptome profiles of Arabidopsis plants overexpressing homologous ADC2,SAMDC1, or SPMS shows a preferential induction of stress-related genes[65–67]. When the expression profiles of those Put and Spm overproducer plantsare compared, a set of 71 genes always appear to be upregulated and enriched instress-related genes, including Ca2þ signaling-related proteins, as well as severalputative transcription factors [66].

27.4 Possible Mechanisms of PA Action in Stress Responses j629

The existence of plant PAmodulon expression system, in a situation similar to thesystems proposed previously in Escherichia coli [68] and yeast [69], could be one of thepossible explanations for some of the transcriptional changes observed in PAaccumulating plants, although the identification of members of this plant PAmodulon is still an issue unresolved. In the future, an additional effort in the isolationof the transcription factors controlling expression of genes when PA metabolism ismodified is needed to clarify the possible existence of a �PA plant modulon.�

Also, most of the transcriptional changes observed could be a consequence ofcrosstalk of PAs and other signaling routes [70]. There are some evidences ofcrosstalk between PAs and ABA. Upregulation of PA biosynthetic genes ADC2,SPDS1, and SPMS and accumulation of Put under drought stress in Arabidopsis aremainly ABA-dependent responses [34]. Evidence of crosstalk with Spm and ABA isalso shown in SAMDC1 overexpressing Arabidopsis plants, with elevated levels ofABAdue to the induction ofNCED3, a key enzyme involved inABAbiosynthesis [67].

Apossible link between PAs, Ca2þ homeostasis, and stress responses has also beenpointed out [56]. Spm control of Ca2þ allocation through regulating Ca2þ permeablechannels, including CAXs, has been described as a possible way of action for theprotective role of Spm against high salt and drought stress [13, 71].Moreover, changesin free Ca2þ in the cytoplasm of guard cells are involved in stomatal movement thatmay explain drought tolerance induced by Spm.What is more, Ca2þ signaling genesare one of the gene categories mainly upregulated in Put and Spm overproducerplants [66]. Also, a �Spm signaling pathway� has been proposed to explain the role ofenhancedPAaccumulationobservedduringpathogenresponse inArabidopsis [72, 73].This signaling pathway could function via the merged signal of Spm-activated Ca2þ

influxandH2O2producedbySpmdegradationforPAoxidases.Bothprocesses areableto trigger mitochondrial dysfunction and activation of the cell death programme [73].

Put, Spd, and Spm also regulate stomatal responses by reducing their aperture andinducing closure [74, 75]. It has been proposed that PAs could regulate stomatalclosure in amechanism involving peroxide productionbypolyamineoxidation, aswellas interactions with nitric oxide (NO) signaling [76]. Thus, PAs could act synergicallywith reactive oxygen species (ROS) and NO in promoting ABA responses in guardcells [56]. PAs could also regulate stomatal closure responses by their capacity to blockfast-activating vacuolar cation channels by their charge properties, as well as affectingprotein kinase and phosphatase activities that regulate ion channel functions [56].

In summary, PA action in plant stress responses seems to imply several layers ofaction. Figure 27.2 illustrates the possible mechanisms underlying enhanced stresstolerance shown by plants with enhanced PA production obtained by transgenicmodification. In conclusion, manipulation of polyamine metabolism seems to be agood strategy to obtain tolerant plants, both by the protecting role that PA can exert bytheir structure and by their capacity to act as a key regulatory molecule in stressresponses. Combination of both factors could lead to improved stress tolerance inplants (Figure 27.2).

All these studies demonstrated that a transgenic approach involving PA biosyn-thetic genes may be a good strategy to improve crop tolerance against harshenvironments so as to meet the requirements of a challenging global environment.

630j 27 Polyamines in Developing Stress-Resistant Crops

Interestingly, broad-spectrum tolerance (salinity, drought, low and high temperature,and parquet toxicity) is observed for some of the transgenic plants overexpressingADC, SPDS, or SAMDC (Table 27.1). Such multiple abiotic stress tolerance is ofpractical importance since plants often suffer from several concurrent forms ofenvironmental stress during their life cycle.

Acknowledgment

Thework in the laboratory ofNT is partially supported by the grants fromDepartmentof Biotechnology and Department of Science and Technology, Government of India.

Figure 27.2 Possible mechanisms underlyingenhanced stress tolerance via transgenicalteration of polyamine biosynthetic genes.Overexpression of PA biosynthetic genes leadsto changes in PA levels in plants (accumulationof individual or total PAs) that could act, on theone hand, as antioxidants to scavenge excessivefree radicals or asmembrane stabilizers through

binding to structures with negatively chargedgroups, such as DNA, proteins, and lipids, andon the other hand, being able to activate plantdefense response mechanisms directly (byunknownmechanisms) and by interactionswithother signaling pathways, such as ABA, Ca2þ , orNO. Combination of these factors could lead toplant stress tolerance. Adapted from Ref. [33].

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