OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice ( Oryza sativa L. cv. IR64)

OsSUV3 dual helicase functions in salinity stress tolerance bymaintaining photosynthesis and antioxidant machinery inrice (Oryza sativa L. cv. IR64)

Narendra Tuteja*, Ranjan Kumar Sahoo, Bharti Garg and Renu Tuteja

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

Received 28 February 2013; revised 17 June 2013; accepted 24 June 2013.

*For correspondence (e-mails [email protected]; [email protected]).

Accession number: GQ982584

SUMMARY

To overcome the salinity-induced loss of crop yield, a salinity-tolerant trait is required. The SUV3 helicase is

involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of

plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the

Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we

report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a

constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance.

The T1 and T2 sense transgenic lines showed tolerance to high salinity and fully matured without any loss

in yields. The T2 transgenic lines also showed tolerance to drought stress. These results suggest that the

introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines

showed lesser lipid peroxidation, electrolyte leakage and H2O2 production, along with higher activities of

antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense trans-

genic lines. These results suggest the existence of an efficient antioxidant defence system to cope with

salinity-induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA heli-

case and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance

without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress

tolerance by improving photosynthesis and antioxidant machinery in transgenic rice.

Keywords: antioxidant, ATPase, DNA and RNA helicase, Oryza sativa, salinity stress, SUV3, unwinding.

INTRODUCTION

Abiotic stresses represent the most limiting environmental

factors affecting agricultural productivity. To overcome

these limitations and to improve production in order to

feed the ever-increasing population, it is imperative to

develop crop cultivars that are stress tolerant. Soil salinity

and drought stress are increasing threats for agriculture;

therefore, it is necessary to develop stress-tolerant varie-

ties (Mahajan and Tuteja, 2005; Tuteja, 2007a,b). Many

genes including helicases are known to be involved in

abiotic stress tolerance. Helicases are ubiquitous enzymes

that catalyse the unwinding of energetically stable duplex

DNA or RNA secondary structures, and thereby play an

important role in almost all DNA and/or RNA metabolic

processes, including replication, DNA repair, recombina-

tion, transcription, pre-mRNA processing, RNA degrada-

tion and translation (Tuteja, 2003; Tuteja and Tuteja, 2004;

Abdelhaleem, 2010). Based on several conserved amino

acid sequence motifs present in helicases, they are classi-

fied into five different superfamilies (SFs), designated

SF1–SF5 (Gorbalenya and Koonin, 1993). SF1 and SF2 are

the largest, and their members contain nine conserved

motifs (Q, I. Ia, Ib, II, III, IV, V and VI) that constitute the

helicase core region (approximately 350–700 amino acids).

Based on variations in motif II, the SF2 family of helicases

is further divided into subgroups: DEAD box, DEAH

box, and Ski2-like proteins, generally referred to as DExD/

H box helicases (Tuteja and Tuteja, 2006; Umate et al.,

2010). All these helicase conserved motifs are located in

two different domains: domain 1 contains motifs Q–III,

whereas domain 2 contains motifs IV–VI (Bleichert and

Baserga, 2007). The Q motif is present upstream of motif I,

and consists of an invariant glutamine (Q) in a sequence of

nine amino acids, and is therefore given the name ‘Q

motif’. The functions of these motifs have been described

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd

1

The Plant Journal (2013) doi: 10.1111/tpj.12277

earlier (Tuteja and Tuteja, 2006; Bleichert and Baserga,

2007). Helicases are also involved in responses to abiotic

stress (Vashisht and Tuteja, 2006; Owttrim, 2013). Earlier, a

Pisum sativum (pea) helicase (PDH45) was reported to be

induced by salinity stress, and was shown to be involved

in salinity tolerance in transgenic Nicotiana tabacum

(tobacco; Sanan-Mishra et al., 2005) and Oryza sativa (rice;

Amin et al., 2012; Sahoo et al., 2012).

The SUV3 (suppressor of Var 3) gene encodes an NTP-

dependent DNA/RNA helicase that belongs to the DExH/D

(Ski2p) superfamily. The SUV3 helicase was originally

identified in Saccharomyces cerevisiae (yeast) as a domi-

nant suppressor allele, SUV3–1, that suppressed three

dodecamer deletion phenotypes on the VAR1 gene (Butow

et al., 1989). The product of the nuclear-encoded SUV3

gene in S. cerevisiae was reported to be localized in mito-

chondria, and is a subunit of the degradosome complex

that regulates RNA surveillance and turnover (Dziembow-

ski et al., 2003; Malecki et al., 2007). In humans, hSuv3p

has been shown mainly in the mitochondrial matrix, and is

essential for the degradation of mature mtRNAs (Szczesny

et al., 2010). hSuv3p unwinds double-stranded DNA,

double-stranded RNA and RNA-DNA heteroduplexes (Shu

et al., 2004). Yeast SUV3 was reported to be involved in

mtDNA replication, maintenance of mtDNA stability and

RNA turnover (Guo et al., 2011). To date, plant SUV3 has

not been characterized in detail. Gagliardia et al. (1999)

have reported that nuclear-encoded Arabidopsis thaliana

SUV3 (AtSUV3) is localized in Arabidopsis mitochondria,

and possesses ATPase activity. Here, we report on the

detailed characterization of SUV3 from rice. Our results

show that the rice SUV3 (OsSUV3) exhibits ATPase, RNA

and DNA helicase activities, and its overexpression in IR64

rice enhances salinity stress tolerance by improving the

antioxidant machinery of the transgenic rice.

RESULTS

Identification and sequence analysis of OsSUV3

OsSUV3 encodes an NTP-dependent RNA/DNA helicase,

which is related to the DExH/D (Ski2p) superfamily. An

alignment of the complete amino-acid sequence of OsSUV3

orthologue with SUV3 from A. thaliana, Homo sapiens and

S. cerevisiae was performed using CLUSTAL W2 (http://www.

ebi.ac.uk). OsSUV3 demonstrates approximately 32–61%

identity with its counterparts from S. cerevisiae, H. sapiens

and A. thaliana (Figure S1). OsSUV3 contains all the char-

acteristic conserved helicase motifs from I, Ia, Ib, II, III, IV, V

and VI (Figure S1). Although there are significant differ-

ences in the sequences of these motifs from other SF2 heli-

cases, some important residues are found to be conserved

among the whole family. For example, the OsSUV3 does

not contain the Q motif, and instead of PTRELA (motif Ia),

DEAD (motif II) and SAT (motif III), it has PLRLLA, DEIQ and

GDP, respectively. The analysis of its amino acid sequence

further indicated that the core region is highly conserved

and that OsSUV3 is smaller in size, compared with its coun-

terparts from S. cerevisiae and H. sapiens (Figure S1). This

difference results from shorter N- and C–terminal regions

in OsSUV3 and AtSUV3, compared with its human and

yeast counterparts (Figure S1). Further detailed analysis of

the protein sequence at Expasy (http://prosite.expasy.org)

indicated that both OsSUV3 and AtSUV3 contain two dis-

tinct domains: a helicase ATP-binding domain and a

helicase C–terminal domain (Figure S2a,b).

Molecular modelling of OsSUV3 structure and secondary

structure analysis

For structural modelling, the sequence of full-length

OsSUV3 was submitted to the Swiss Model homology-

modelling server (http://swissmodel.expasy.org) (Arnold et al.,

2006). The model that was built using H. sapiens SUV3 as

the template was studied in detail (Jedrzejczak et al., 2011).

OsSUV3 primary sequence residues 59–541 showed

approximately 40% sequence identity with the SUV3 heli-

case from H. sapiens (Jedrzejczak et al., 2011). The struc-

tural modelling of OsSUV3 was therefore performed using

the known crystal structure of this homologue as the tem-

plate (Protein Data Bank (PDB) number 3rc8A at http://

www.rcsb.org/pdb). The ribbon diagram of the template is

shown in Figure 1a, and the predicted structure of OsSUV3

is shown in Figure 1b. When the modelled structure of

OsSUV3 and the template were superimposed, it is clear

that these structures superimpose partially (Figure 1c).

Molecular graphic images were produced using the UCSF

Chimera package (http://www.cgl.ucsf.edu/chimera) from

the Resource for Biocomputing, Visualization and Informat-

ics at the University of California, San Francisco (supported

by NIH P41 RR-01081; Pettersen et al., 2004). The PDB file

of the modelled OsSUV3 protein was subjected to the PDB-

sum server (http://www.ebi.ac.uk/thornton-srv/databases/

pdbsum/Generate.html) for further secondary structure

analysis (Laskowski, 2009). The predicted secondary struc-

ture of the OsSUV3 protein shows the presence of four

sheets, three b–a–b units, two b hairpins, one b bulge,

18 strands, 22 helices, 27 helix–helix interactions, 35

b turns and three c turns (Figure S2c).

Purification and characterization of OsSUV3

The OsSUV3 cDNA was expressed in Escherichia coli, add-

ing a six-histidine tag at its C terminus. The approximately

67–kDa OsSUV3 protein was purified to near homogeneity

and confirmed by SDS-PAGE analysis (Figure 1d, lane 2).

The identity of the purified protein was confirmed by wes-

tern blot analysis using anti-His antibody (Figure 1e,

lane 2). This purified preparation was used for all of the

enzyme assays. The ssDNA-dependent ATPase activity

of OsSUV3 protein was checked using standard assay

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277

2 Narendra Tuteja et al.

conditions, as described in the Experimental procedures,

in the presence of traces of radiolabelled ATP with 1 mM

cold ATP and purified enzyme (10 ng). OsSUV3 protein

(10 ng) exhibits ATPase (Figure 1f, lane 2), DNA unwinding

(Figure 1g, lane 3) and RNA helicase activities (Figure 1h,

lane 2).

Expression profile of the OsSUV3 gene in wild-type IR64

rice in response to abiotic stress

The salt treatment of IR64 rice seedlings showed a signifi-

cant increase in the transcript level of OsSUV3. The

200–mM NaCl treatment induced a roughly fivefold increase

in expression of OsSUV3 during the first hour (1 h), and

this transcript accumulation gradually increased until 12 h

(approximately 13-fold; Figure 2a). It appears as an early as

well as prolonged and strong response against NaCl expo-

sure. However, as compared with the NaCl, the WT plants

accumulated lesser transcripts of OsSUV3 when subjected

to KCl treatment (Figure 2b). The maximum expression of

OsSUV3 was five-fold after treatment with KCl (Figure 2b; 2

and 12 h), as opposed to a 13–fold increase after NaCl treat-

ment. The heat stress upregulated the OsSUV3 transcript

level to a lesser extent (threefold at 2 and 12 h), as com-

pared with the NaCl treatment (Figure 2c). ABA treatment

induced OsSUV3 with a sixfold increase in expression

during the early period (2 h; Figure 2d).

Response of T1 transgenic IR64 rice plants to salt stress

The T–DNA construct of the OsSUV3 gene (sense and anti-

sense orientation) used for the development of transgenic

rice plants is shown in Figure 3a. The analysis for the

presence of genomic integration of the transgene was car-

ried out on T1 plants. Phenotypically there were no signifi-

cant differences among the empty vector control (VC),

antisense (AS) and sense lines (L1–L3) of transgenic rice

plants, as compared with the WT plants. The integration of

the transgene (SUV3) was confirmed twice by PCR, and the

observed copy number was one in lines 1 and 2, and two

in line 3, as described earlier (Sahoo and Tuteja, 2012).

The Gus activity was found to be positive in leaf tissues of

all the three transgenic lines (L1–L3), as well as in the AS

and VC plants (Figure 3b).

The quantitative real-time PCR (qRT-PCR) showed

between eight and ninefold induction in the transcript level

of sense transgenic lines (L1–L3), compared with WT plants

under normal (unstressed) conditions (Figure 3c). The

salinity tolerance index of T1 sense transgenic lines was

found to be higher (79.8, 81.6 and 80.8%, respectively) in

comparison with WT plants (33.8%) (Figure 3d). The AS

and VC plants showed the same expression and salinity

tolerance indexes as WT plants.

To further test salinity tolerance, leaf discs from T1 sense

transgenic lines, WT, VC and AS rice plants were floated

separately on 100 and 200 mM NaCl for 96 h. The salinity-

induced loss of chlorophyll was lesser in sense transgenic

lines compared with WT, VC and AS plants (Figure 3e).

The damage caused by stress was reflected in the degree

of bleaching observed in the leaf tissue after 96 h. The

measurement of the chlorophyll content of the leaf discs

from all the above plants provided further evident support

for a positive co-relationship between the T1 sense trans-

genic lines and tolerance of salinity stress (Figure 3f).

(a) (d) (e)

(f) (g) (h)

(b)

(c)

Figure 1. Structure modelling, purification and

enzymatic activities of the OsSUV3 protein.

(a–c) Structure modelling: (a) template; (b)

OsSUV3; (c) superimposed image. (d,e) Purifica-

tion of OsSUV3. (d) Coomassie blue-stained gel

of purified OsSUV3: lane 1, molecular weight

marker; lane 2, purified OsSUV3 (200 ng).

(e) Western blot of purified OsSUV3: lane 1,

protein molecular weight marker; lane 2,

purified OsSUV3. (f–h) Enzymatic activities of

OsSUV3. (f) ATPase activity: lane 1, control

reaction without enzyme; lane 2, reaction with

OsSUV3 (10 ng). (g) DNA helicase activity.

Lane 1, control reaction without enzyme; lane 2,

boiled substrate; lane 3, reaction with OsSUV3

(10 ng); S, substrate; UD is unwound DNA. (h)

RNA helicase activity: lane 1, control reaction

without enzyme; lane 2, reaction with OsSUV3

(10 ng); lane 3, heat-denatured substrate; S,

substrate; UR is unwound RNA.


Rice SUV3 helicase functions in salinity tolerance 3

(a) (b)

(c) (d)

Figure 2. Quantitative RT-PCR analyses of

OsSUV3 under different abiotic stress condi-

tions: (a) 200 mM NaCl; (b) 200 mM KCl; (c) heat

stress at 45°C; (d) 100 lM ABA. Error bars indi-

cate the standard errors (�SEs) calculated from

three independent experiments. Different letters

on top of the bars indicate significant differ-

ences at a level of P < 0.05, as determined by

Duncan’s multiple range test (DMRT).

(a) (b)

(c) (d)

(e) (f)

Figure 3. Analysis and expression of T1 trans-

genic lines (OsSUV3). (a) OsSUV3 gene cloned

in pCAMBIA1301 vector at HindIII site. (b) Histo-

chemical GUS assay shows the expression of

GUS gene (blue stain) in the SUV3 transgenics

leaves. (c) Relative expression of the OsSUV3

gene in WT and transgenic lines under control

(unstressed) conditions. (d) Salinity tolerance

index of T1 sense transgenic lines, as compared

with WT, VC and AS plants. (e) Leaf disc senes-

cence assay for salt tolerance in T1 OsSUV3

transgenic rice lines, as compared with WT, VC

and AS plants under 100 and 200 mM NaCl

concentrations. (f) Chlorophyll content (mg per

g fresh weight) in T1 OsSUV3 transgenic lines

under 100 and 200 mM NaCl. In panels (b–f), WT

is wild type, VC is the empty vector control, AS

is antisense, and L1–L3 are the sense transgenic

lines. Error bars in (c), (d) and (f) panels indicate

standard errors (�SEs) calculated from three

independent experiments. Different letters on

top of the bars indicate significant differences

at a level of P < 0.05, as determined by

Duncan’s multiple range test (DMRT).



OsSUV3 T1 sense transgenic rice plants accumulate less

MDA, H2O2 and ion leakage, and show better antioxidant

response

We compared the salt-induced changes in the accumula-

tion of H2O2, MDA (lipid peroxidation product) and ion

leakage in T1 sense transgenic lines (L1–L3), WT and VC

rice seedlings. The MDA, H2O2 and ion leakage levels

were significantly reduced in OsSUV3 sense transgenic

lines under salt stress (200 mM NaCl), as compared with

WT and VC seedlings (Figure 4a–c). These results indicate

that overexpression of OsSUV3 could decrease the accu-

mulation of reactive oxygen species (ROS) in sense trans-

genic rice seedlings. The data for AS seedlings were

found to be almost similar to those for the WT and VC

seedlings.

Salt treatment (200 mM NaCl) increased the activities of

CAT, APX and GR (Figure 4d–f) in both WT and transgenic

plants; however, the OsSUV3 sense transgenic lines

(L1–L3) exhibited a higher increase in the activities of anti-

oxidant enzymes (except CAT), as compared with WT and

VC seedlings in response to salt stress. Proline accumula-

tion was strongly upregulated in OsSUV3 T1 sense trans-

genic lines (Figure 4g), which eventually also maintained

the water balance (Figure 4h) in these lines during salt-

stress conditions. The data for AS seedlings were almost

similar to those for the WT and VC seedlings.

Agronomic performance of T1 transgenic plants

There was no significant difference observed in the sur-

vival rates of seedlings of the T1 sense transgenic (with

0

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Figure 4. Biochemical analysis of T1 OsSUV3 transgenic lines (L1–L3) under conditions of 200 mM NaCl. (a) Determination of lipid peroxidation expressed in

terms of Malondialdehyde (MDA) content in OsSUV3 transgenic lines, WT and VC. (b) Changes in the level of hydrogen peroxide (H2O2) content in OsSUV3

transgenic lines, WT and VC. (c) Estimation of the percentage electrolytic leakage in OsSUV3 transgenic lines, WT and VC. (d) Catalase (CAT) activity in OsSUV3

transgenic lines, WT and VC. One unit of enzyme activity is defined as 1 lmol H2O2 oxidized per min. (e) Changes in ascorbate peroxidase (APX) enzyme activity

in OsSUV3 transgenic lines, WT and VC. One unit of enzyme activity is defined as 1 lmol of ascorbate oxidized per min. (f) Changes in glutathione reductase

(GR) enzyme activity in OsSUV3 transgenic lines, WT and VC. One unit of enzyme activity is defined as 1 lmol of glutathione synthetase-5-thionitrobenzoic acid

(GS-TNB) formed per min as a result of the reduction of 5-5’-dithiobis (2-nitrobenzoic acid) (DTNB). (g) Changes in the level of proline accumulation in OsSUV3

transgenic lines, WT and VC. (h) Estimation of the percentage relative water content (RWC) in OsSUV3 transgenic lines, WT and VC. Data represent the

means � SDs of three independent experiments (n = 3), aP < 0.05, bP < 0.01, cP < 0.001.



NaCl stress), as compared with seedlings of WT, VC and

AS (without stress) (Table 1). The seeds showing hygromy-

cin resistance clearly displayed a segregation ratio of 3:1 in

inoculation analysis (Table 1). Significant differences in

growth parameters were observed between WT and T1

sense transgenics (under 200 mM NaCl stress) lines. The

OsSUV3 sense transgenic plants showed better perfor-

mance in several growth parameters, such as plant height,

root length, root dry weight and leaf area, under salt

stress, as compared with the WT (Table 2). Several yield

attributes, such as days required for flowering, number of

tillers per plant, panicles per plant, filled grain per panicle,

chaffy grain per panicle, 100 grain weight at 200 mM NaCl

were recorded and found to be almost similar to the WT

plants grown in water (0 mM NaCl). However, the WT

plants did not survive till flowering stage under 200 mM

NaCl stress (Table 3). Under identical conditions the

growth parameters and yield attributes of VC and AS

plants were almost similar to WT plants.

Measurement of photosynthetic characteristics

Photosynthetic machinery was also severely affected by

salt stress, but the extent of the damage was higher in the

WT as compared with sense transgenic plants (Table 2).

OsSUV3 T1 sense transgenic lines experienced less reduc-

tion in chlorophyll content and total protein content, com-

pared with WT plants, under 200 mM NaCl stress. OsSUV3

sense transgenic plants showed a lesser percentage reduc-

tion in net photosynthetic rate, in comparison with WT

plants. Moreover, stomatal conductance and intercellular

CO2 also followed the same higher trend as the net photo-

synthetic rate in transgenic lines, compared with WT plants

(Table 2). Under similar conditions the photosynthetic

characters of VC and AS plants were similar to those in WT

plants.

Estimation of endogenous ion contents

Salt-treated T1 sense transgenic lines showed more accu-

mulation of nitrogen, phosphorus and potassium, and less

accumulation of sodium, in comparison with WT plants

(Table 2). All the plants (WT and sense) contain almost the

same nutrients when compared with conditions of no

stress (0 mM NaCl). Under similar conditions the endo-

genous ion contents of VC and AS plants were almost

identical to that of WT plants.

Analysis and confirmation of T2 transgenic IR64 rice plants

and their response to salt stress

The integration of transgene and different phenotypic char-

acters were studied in OsSUV3 T2 sense transgenic lines.

Phenotypically the T2 sense transgenic plants were similar

to the WT, VC and AS plants. The integration of the

OsSUV3 gene (1.7 kb) was confirmed by PCR in all the

transgenic lines using gene-specific primers (Figure 5a).

The amplification of the transgene was further confirmed

by using promoter-specific (CaMV 35S) forward and gene-

specific reverse primers, and the expected size (2.2–kb)

fragment was obtained (Figure 5b). The qRT-PCR showed

a between seven- and ninefold induction in the transcript

level of T2 sense transgenic lines (L1–L3), as compared

with WT plants under normal (unstressed) conditions

(Figure 5c). GUS activity was visualized in the leaf tissue of

all three transgenic lines of T2 plants, and they all showed

expression of the GUS gene but the WT plants were not

GUS-positive (Figure 5d).

To study the effect of salt stress during germination,

seeds of WT and T2 transgenic plants were grown on MS

plates (Murashige and Skoog, 1962) supplemented with

200 mM NaCl. The sense transgenic seeds showed efficient

growth, whereas lesser or no germination was observed in

the case of WT seeds under salt stress (Figure 5e). The VC

and AS plants showed germination patterns similar to that

of WT plants. In the leaf disc assay the salinity stress-

induced loss of chlorophyll was lower in OsSUV3 T2 sense

transgenic lines, as compared with WT, VC and AS plants

(Figure 5f). The damage caused by stress was visible in the

degree of bleaching observed in the leaf-disc tissue after

96 h. Moreover, measurement of the chlorophyll content

supported the leaf disc assay results under 100 and

200 mM NaCl stress (Figure 5g).

T2 sense transgenic plants showed better growth perfor-

mance under salt stress. The leaves of control (AS, VC and

WT) plants showed curling and dropping characteristics

during the initial period of stress (after 2 days of salt

Table 1 Comparison of segregation ratio and plant seedling survival (%) of the WT and T1 generation of OsSUV3 overexpressing transgenicplants (lines 1–3; Oryza sativa L. cv. IR64) grown in the presence of 0 (H2O) or 200 mM NaCl, respectively

Attributes

Water-grown control plants 200 mM NaCl-grown OsSUV3 transgenic plants

WT VC AS Line 1 Line 2 Line 3

Segregation ratio 0 3.1:1 (132) 3.2:1 (132) 3.3:1 (152) 2.85:1 (156) 3.2:1 (142)Plant seedling survival (%) 98 � 3.8a 98 � 3.8a 97 � 3.8a 98 � 4.1a 98 � 3.8a 97 � 4.2a

Each value represents the mean of three replicates � SEs.AS, antisense transgenics; VC, vector control transgenics; WT, wild type.The letters a, b, c indicate significant differences at the level of P > 0.05, as determined by Duncan’s multiple range test (DMRT).



Table

2Growth

[plantheight(cm),

rootlength

(RL),rootdry

weight(g),

leaf

area

(cm

2)],photosy

nthes

is[totalch

lorophyllco

ntent(m

gper

gfres

hweight);net

photosy

nthetic

rate

(lmolCO

2m

�2s�

1),stomatal

conductan

ce(m

molm

�2s�

1)an

dinternal

CO

2co

nce

ntration(lmolmol�

1)an

dtotalprotein

(mgper

gfres

hweight)]an

dnutrients

[nitrogen

(%),phosp

horus

(%),potassium

(%),so

dium

(%)]ofnon-transg

enic

(WT)an

dT1gen

erationofOsS

UV3ove

rexp

ressingtran

sgen

iclin

es(lines

1–3)

ofrice

(Oryza

sativa

L.cv

.IR64

)grownwith0or20

0m

M

NaC

l

Attributes

ControlWTplants

200m

MNaC

l-grownT1OsS

UV3tran

sgen

icplants

0m

MNaC

l20

0m

MNaC

l

Line1

Line2

Line3

0m

MNaC

l20

0m

MNaC

l0m

MNaC

l20

0m

MNaC

l0m

MNaC

l20

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treatment; Figure 5h); however, after 12 and 30 days of salt

treatment, the SUV3 sense plants (L1) survived more effi-

ciently up to maturity, and gave viable seeds (Figure 5i,j),

whereas the control (AS, VC and WT) plants completely

died. The other two T2 sense transgenic lines (L2 and L3)

showed similar performances as L1 under salt stress.

Effect of drought and cold stress on post-germination

growth of T2 transgenic seeds

Seeds of T2 sense transgenic plants showed good post-

germination growth under drought stress conditions,

whereas WT seeds failed to germinate under the same con-

ditions (Figure 6a). There was no germination in T2 trans-

genics and WT seeds at 4°C, for up to 14 days (Figure 6b).

Interactome of OsSUV3

The results of interactome analysis showed that OsSUV3

interacts with a variety of different proteins, such as exori-

bonuclease, exonuclease, endonuclease, some splicing

factors, and a few RNA and DNA helicases (Figure S3).

DISCUSSION

Helicases are evolutionarily conserved proteins that are

ubiquitous in nature, and are known to be involved in

diverse cellular and metabolic processes, including their

new emerging role in plant abiotic stress tolerance (Vash-

isht and Tuteja, 2006; Tuteja, 2007a; Umate et al., 2010;

Owttrim, 2013). The OsSUV3 gene encodes a DNA/RNA

helicase and belongs to the family of DExH-box helicases.

In the present study we have characterized the SUV3

homologue from O. sativa. This study shows that OsSUV3

protein contains the highest sequence homology to A. tha-

liana SUV3 mitochondrial helicase, as compared with its

yeast and human counterparts. Similar to both yeast and

human counterparts, AtSUV3 is also present in the mito-

chondria (Gagliardia et al., 1999); therefore, it is most likely

that OsSUV3 is also present in mitochondria.

Both OsSUV3 and AtSUV3 exhibit the characteristic heli-

case ATP-binding and helicase C–terminal domains, with

some peculiarities and uniqueness in the sequences of the

conserved motifs, which are almost similar to the human

SUV3 (Jedrzejczak et al., 2011). In OsSUV3 there is no

Q motif, and DEIQ is present instead of DEAD. Although

most of the typical helicase motifs are present in OsSUV3,

but the conserved sequences show some unique character-

istics, suggesting that OsSUV3 protein may constitute a

separate subfamily of helicases, as also suggested for

human SUV3 helicase (Jedrzejczak et al., 2011). OsSUV3

protein contains ATPase and DNA and RNA helicase activi-

ties, which is similar to its human counterpart (Shu et al.,

2004). To the best of our knowledge the DNA and RNA

helicase activities in an SUV3 homologue from plant spe-

cies have not been reported so far. In the case of yeast

SUV3, the point mutants K245A and V272L carrying muta-

tions in the helicase motifs I and Ia, respectively, showed

the involvement of SUV3 in RNA turnover andmtDNAmain-

tenance (Guo et al., 2011). As these mutations abolish the

ATPase and helicase activities of the yeast SUV3 protein,

these results also confirm, therefore, that the biochemically

active protein is required for the functions of the protein.

Table 3 Comparison of various yield parameters of non-transgenic (WT) and T1 generation of OsSUV3 overexpressing transgenic lines(lines 1–3) of rice (Oryza sativa L. cv. IR64) grown with 0 or 200 mm NaCl, respectively

Yield attributes

Control WT plants 200 mM NaCl-grown T1 OsSUV3 transgenic plants

0 mM NaCl200 mM

NaCl

Line 1 Line 2 Line 3

0 mM NaCl 200 mM NaCl 0 mM NaCl 200 mM NaCl 0 mM NaCl 200 mM NaCl

Time required forflowering (days)

90 � 2.5a ND* 93 � 3.8a 90 � 2.5a 92 � 3.2a 90 � 2.6a 93 � 3.5a 90 � 2.5a

No. of tillers perplant

26 � 1.0c ND 33 � 0.13a,b 31 � 1.2a,b 31 � 0.14a,b 31 � 1.1a,b 39 � 0.12a 37 � 1.0a

No. of paniclesper plant

22 � 0.7c ND 30 � 0.12a,b 28 � 1.0a,b 33 � 0.12a,b 29 � 1.0a,b 37 � 0.15a 35 � 1.1a

No. of filled grainsper panicle

70 � 3.2b ND 103 � 4.81a 98 � 4.1a 105 � 4.63a 97 � 4.3a 108 � 4.77a 98 � 4.1a

No. of chaffy grainsper panicle

12 � 0.33a ND 07 � 0.11b 05 � 0.21b 03 � 0.07b 05 � 0.20b 04 � 0.23b 07 � 0.11b

Straw dry weight (g) 53 � 2.1b ND 69 � 3.05a 65 � 2.5a 70 � 3.13a 67 � 2.1a 70 � 2.9b 71 � 2.6a

100-grain weight 2.81 � 0.1a ND 2.89 � 0.130b 2.83 � 0.12a 2.87 � 0.122b 2.83 � 0.10a 2.86 � 0.14b 2.83 � 0.11a

ND, no data.*WT plants did not survive until harvest under 200 mM NaCl.Each value represents the mean of three replicates � SE.Means were compared using ANOVA.Data followed by the same letters in a row are not significantly different at P > 0.05, as determined by the least-significant difference (LSD)test. a,b,cSignificant differences at the level of P > 0.05, as determined by Duncan’s multiple range test (DMRT).



The transcript level of the OsSUV3 gene was found to be

induced by several fold in response to NaCl, as compared

with KCl. An Na+–specific response has previously been

reported for the PDH45 gene (Sanan-Mishra et al., 2005).

The heat stress had no effect on the expression of the

OsSUV3 gene. The transcript of the OsSUV3 gene was also

found to be induced in response to the phytohormone, ABA,

which is already known for activation and repression under

multiple stress conditions (Tuteja, 2007b). Similar to Os-

SUV3 the transcript of the PDH45 gene was also reported to

be induced in response to ABA (Sanan-Mishra et al., 2005).

Rice plants expressing the OsSUV3 gene show enhanced

tolerance to salinity stress, as indicated by the higher chlo-

rophyll content, photosynthesis and plant dry weight of

NaCl-stressed transgenic plants in comparison with WT

plants. Moreover, the T1 as well as T2 rice seedlings were

able to grow, flower and set viable seeds under continuous

NaCl stress. This result suggests that the introduced trait is

functional and stable in transgenic rice plants. Interestingly,

the NaCl-stressed rice transgenics showed yield stability,

because there was no loss in seed number. The transgenic

lines accumulated lesser quantities of Na+ than theWT plants.

Lower Na+ content in the leaves of OsSUV3-expressing lines

of rice plants showed less damage to photosynthetic appa-

ratus, thus maintaining normal growth and plant dry weight

and yield, whereas WT plants accumulated higher Na+ con-

(a)

(e) (f) (g)

(h) (i) (j)

(b) (c) (d)

Figure 5. T2 SUV3 transgenic lines were used for further analysis. (a) Confirmation of SUV3 overexpressing sense lines (L1–L3) by PCR using gene-specific

forward and reverse primers. (b) PCR analysis of T2 OsSUV3 sense transgenic plants by using the promoter (CaMV 35S) forward and OsSUV3 gene-specific

reverse primers. (c) Relative expression of OsSUV3 gene in WT and T2 sense transgenic lines under unstressed conditions. (d) GUS assay of OsSUV3 T2 trans-

genic lines. (e) Germination of T2 OsSUV3 transgenic and WT seeds on an MS plate supplemented with 200 mM NaCl. (f) Leaf disc assay of T2 OsSUV3 trans-

genic rice under salinity stress (100 and 200 mM NaCl). (g) Chlorophyll estimation (mg per g fresh weight) of WT and T2 transgenics under 100 and 200 mM

NaCl. (h) Salt tolerance response of transgenic plants [sense OsSUV3, antisense (AS) OsSUV3, VC (empty vector-pCAMBIA1301)] and WT after 2 days of 200 mM

NaCl stress. (i) Salt stress tolerance response of same set of plants after 12 days of 200 mM NaCl stress. (j) Salt stress tolerance of same set of mature plants

after 30 days of NaCl stress.



tent and experienced damage. The inhibition of photosyn-

thesis under salinity stress may be attributed to stomatal

closure caused by water deficit, in addition to several other

biochemical and photochemical processes, like imbalance

between ROS and antioxidant machinery.

Increased ROS produced during salt stress can cause

damage to cellular macromolecules, thus causing MDA

accumulation, which ultimately affects the stability of

membranes (Apel and Hirt, 2004; Gill and Tuteja, 2010; Gill

et al., 2012). OsSUV3 sense transgenic lines showed lesser

lipid peroxidation, ion leakage and H2O2 production, along

with increased activities of antioxidant enzymes (CAT, APX

and GR), which is in tune with other previously reported

studies (Apel and Hirt, 2004). The efficient scavenging

activity of ROS in OsSUV3 sense transgenic lines mini-

mizes the damage to macromolecules, and thus prevents

membrane damage, for the survival of the plant. These

findings are in agreement with earlier studies reported in

another variety of transgenic rice overexpressing PDH45

(O. sativa L. cv. PB1; Gill et al., 2013). The higher proline

accumulation in OsSUV3 T1 transgenic lines probably

provides protection against the ROS-induced disruption of

lipid content of the membranes, resulting in membrane

stability for the survival of plant. The presence of a number

of interacting partners of OsSUV3 suggests that this

enzyme might be involved in diverse cellular activities,

which lead to the observed salinity tolerance. The exact

mechanism of helicase-mediated salinity tolerance is not

yet understood. Most probably OsSUV3 is helping in salt

tolerance by improving the antioxidant machinery and by

maintaining mitochondrial genome integrity of the trans-

genic rice plants under salt stress conditions.

Although the exact mechanism is not known yet, the

interactome analysis of OsSUV3 revealed that it might be

involved in a number of pathways that cumulatively result

in imparting salinity stress tolerance. Overall, the mainte-

nance of better water balance, higher accumulation of

osmo-protectant and enhanced activities of antioxidant

enzymes protect the OsSUV3 sense transgenic lines from

the deleterious effects of oxidative damage, thus contribut-

ing effective tolerance to salt stress. From these observa-

tions, we can conclude that the upregulation of ROS

machinery could be one of the main mechanism for

providing salt tolerance in OsSUV3 transgenic lines.

In plant organelles, including mitochondria, some hair-

pin structures are present at the 3′ termini of the transcripts

needed for processing mRNA and RNA degradation to

regulate gene expression (Gagliardia et al., 1999). These

hairpin structures have been reported to be increased or

misfolded during environmental stress (Vashisht and Tuteja,

2006; Tuteja, 2007a; Kang et al., 2013; Owttrim, 2013). The

functions of RNA helicases are more prominent after the

cells are exposed to stresses, because misfolded RNAs

cannot turn back to native conformation without the help

of RNA helicases. The interactome analysis suggests that

OsSUV3 might be functioning in more than one pathway

in the mitochondria. On the basis of the studies reported, a

supportive hypothetical mechanism could be that OsSUV3

alone, or with the help of predicted mitochondria-localized

interacting partners, probably follows the same pathway in

modulating stem-loop structures during stress conditions

in plants. OsSUV3 might also be playing a role in maintain-

ing mitochondrial genome stability under stress condi-

tions. It will be interesting to characterize OsSUV3 and its

interacting partners in detail to understand its exact mech-

anism in imparting salinity stress tolerance.

EXPERIMENTAL PROCEDURES

Cloning of the rice SUV3 gene

The complete coding region of the 1.74–kb rice SUV3 gene wasPCR amplified by using a forward primer (5′–GGATCCATGGCGTGGCTGCG–3′, with the BamHI site underlined) and a reverseprimer (5′–GGATCCTTTTGATCT CACATCAATTTCTTG–3′, with theBamHI site underlined) designed from the gene sequence, andrice cDNAs as a template. The amplified fragment was cloned intopGEMT easy vector and sequenced (GenBank accession number:GQ982584).

Expression and purification of the rice SUV3 protein

The specific 1.74–kb fragment was excised from pGEMT-OsSUV3plasmid and cloned into the pET28a+ expression vector (Novagen,

(a) (b)Figure 6. (a) Germination of T2 OsSUV3 trans-

genic and WT seeds on an MS plate supple-

mented with 20% polyethylene glycol (PEG).

(b) Germination of T2 OsSUV3 transgenic and

WT seeds on an MS plate at 4°C, for cold stress.



http://www.emdmillipore.com), and the plasmid (pET28a-OsSUV3)was transformed into BL21 (DE3) pLysS cells. A 1% portion of theovernight-grown primary culture was inoculated in 500 ml of Luriabroth (LB) and allowed to grow at 37°C, and the protein wasinduced and purified using Ni-NTA (Qiagen, http://www.qiagen.com) resin and standard protocols. The protein was checked forpurity by SDS-PAGE [10% (w/v) polyacrylamide gel] and coomas-sie staining.

Western blot analysis

The protein was separated by SDS-PAGE and transferred electro-phoretically to nitrocellulose membrane using the standardmethod. After blocking, the membrane was incubated with theappropriate primary antibody (Penta-His; Qiagen) for 3 h at roomtemperature (27�C) and the blot was incubated with the appropri-ate secondary antibody coupled to alkaline phosphatase (Sigma-Aldrich, http://www.sigmaaldrich.com) and developed using thestandard method.

ATPase and helicase assays

The ATPase and DNA and RNA helicase assays were performedwith the purified protein using the method described by Phamet al. (2000).

Plasmid construction and Agrobacterium-mediated

transformation of IR64

The 1.74–kb rice SUV3 gene fragment was cloned in sense andantisense orientation in the pRT100 vector. The CaMV35S-Os-SUV3-polyA fragment thus generated in pRT100 was then insertedinto the multiple cloning site of the rice-compatible pCAMBIA1301containing the hygromycin phosphotransferase-selectable markerto generate the plasmids pCAMBIA1301-OsSUV3 in sense andantisense orientations. A competent strain of Agrobacterium tum-efaciens (LBA4404) was transformed with the sense, antisense(pCAMBIA1301-OsSUV3) and empty vector (pCAMBIA1301) con-struct, as vector control (VC), using standard protocols. The emptyvector contained all except the OsSUV3 gene. Agrobacterium-mediated transformation of IR64 rice was carried out using animproved method (Sahoo and Tuteja, 2012). The VC plants werealso generated at the same time and in the same conditions as theplants containing the vector with the OsSUV3 gene (sense or anti-sense).

PCR, Southern blot analysis and histochemical GUS assay

Integration and the copy number of the OsSUV3 gene was checkedby PCR and Southern blot analysis, as reported previously (Sahooand Tuteja, 2012). Leaves from transgenic (T1 and T2) plants wereconfirmed by b–glucuronidase (GUS) assay (Jefferson, 1987) usingthe indigogenic substrate X–gluc (5–bromo-4-chloro-3-indolylb-D-glucuronide).

RNA isolation and quantitative real-time PCR (qRT-PCR)

Seedlings of the WT (21–day-old O. sativa cv. IR64) were treatedwith 200 mM NaCl, 200 mM KCl, abscisic acid (100 lM ABA) andheat (45°C) under controlled conditions, and samples were har-vested at different time intervals (1, 2, 3, 6 and 12 h). Leaf samplesof unstressed and stressed WT and OsSUV3 T1 transgenic plantswere used for RT-PCR. Total RNA was isolated using TriZOL LSreagent (Invitrogen, http://www.invitrogen.com), following themanufacturer’s instructions, and poly(A)-RNA was isolated. It wasused for making cDNA using the RevertAid H minus first-strand

cDNA synthesis kit (Fermentas, http://www.thermoscientificbio.com/fermentas). Expression analysis of the SUV3 gene was per-formed by qRT-PCR, following the method described by Jayar-aman et al. (2008), and the relative levels of the transcriptaccumulated for the OsSUV3 gene (primers: forward, 5′–CAGTTGAGATGGCCGACA–3′ and reverse 5′–CAGCTGGGTCACCACAAA–3′) were normalized to a–tubulin (primers: forward 5′–GGTGGAGGTGATGATGCTTT–3′ and reverse 5′–ACCACGGGCAAAGTTGTTAG–3′) and OsSUV3 expression in the WT plant (Jain et al.,2006) using the 2–DDCt method from three independent experi-ments (Livak and Schmittgen, 2001). The PCR efficiency, which isdependent on the assay, performance of the master mix and qual-ity of the sample, was calculated as efficiency = 10 (–1/slope) – 1(3.6C slope C 3.1) by the software itself (Applied Biosystems,http://www.appliedbiosystems.com) ‘C’ is defined as thresholdcycle.

Tolerance index (TI)

The TI of the 200 mM NaCl-treated OsSUV3 T1 transgenic (L1–L3)and WT plants were calculated using the following formula: TI(%) = (plant dry weight with 200 mM NaCl)/(plant dry weight withwater) 9 100.

Leaf disc assay for salinity and drought tolerance

The leaf disc assay and chlorophyll measurement were performedas described by Sanan-Mishra et al. (2005).

Determination of antioxidant activities of OsSUV3

transgenic lines

The 21–day-old seedlings of WT and transgenic plants were usedfor biochemical analysis at different time points (1, 6, 12 and 24 h).Estimation of lipid peroxidation, electrolytic leakage, relative watercontent (RWC), measurement of activities of various antioxidantenzymes, including catalase (CAT), ascorbate peroxidase (APX)and glutathione reductase (GR), proline and hydrogen peroxide(H2O2), was performed using the methods described earlier (Garget al., 2012).

Measurement of photosynthetic characteristics

The net photosynthetic rate (PN), stomatal conductance (gs) andintercellular CO2 concentration (Ci) were recorded in fullyexpanded leaves using an infrared gas analyser (IRGA; LI-COR,http://www.licor.com) on a sunny day between 10:00 and 11:00 h.The atmospheric conditions during the measurement were: photo-synthetically active radiation (PAR), 1050 � 7 l mol m�2 s�1; rela-tive humidity, 66 � 4%; atmospheric temperature, 24 � 2°C; andatmospheric CO2, 350 lmol mol�1.

Agronomic performance and estimation of endogenous

ion content of T1 transgenic plants

Growth characteristics were measured at 4 weeks after initiatingthe 0 and 200 mM NaCl treatment in T1 transgenic and WT plants.Shoot and root length was measured on a metre scale. Plant dryweight was determined after drying the samples in an oven at 80°Ctill reaching a constant weight. The leaf area wasmeasured by a leafarea metre (Systronics, Hyderabad, India, http://www.grotal.com/Hyderabad/Systronics-India-Limited-C70/). The total nitrogen con-tent in plant material was determined using the Micro Kjeldahlmethod (Jackson, 1973). The phosphorus content of plant sampleswas calculated as a percentage by using a spectrophotometer(Gupta, 2004). Potassium was estimated via the flame photometer



(Chapman and Pratt, 1982). For the estimation of sodium content,plant material was digested in concentrated HNO3/H2O2 overnight,followed by digestion with 2 M HCl, and analyzed for sodium contentby using simultaneous inductively coupled argon-plasma emissionspectrometry (ICP trace analyzer; Labtam, http://www.labtam-inc.com).

Segregation analysis of the T1 transgenic lines

The inheritance of the OsSUV3 gene in the T1 generation wasanalysed. Here, the progenies were evaluated for resistance tohygromycin. T1 seeds of three independent transformants of theIR64 cultivar were germinated on hygromycin-containing medium(50 mg l�1).

Analysis of T2 transgenic plants

The T2 OsSUV3 transgenic plants were grown to maturity, and theintegration of the transgene was analysed by molecular as well asphenotypic expression in all of the lines, as described for the T1

lines.

Germination test in 200 mM NaCl, 20% PEG and in cold

(4°C) stress

The T2 transgenic rice seeds were germinated at 28°C under200 mM NaCl and 20% PEG for salinity and drought stress, respec-tively. For cold stress, rice seeds (WT and sense lines) were germi-nated in MS medium at 4°C.

Analysis of T2 transgenic plants in the presence of 200 mM

NaCl

The T2 transgenic plants (sense, AS and VC), along with WTplants, were kept together in one big tank filled with 200 mM NaClinstead of water. The response of these plants was recorded at1–day intervals.

Statistical analysis

The experiment was arranged in a randomized block design. Forvarious growth parameters of the WT, VC, AS and OsSUV3 senseT1 transgenic plants, values are presented as means of three repli-cates (each plant was considered a replicate). Here the ‘mean ofthree replicates’ represents the ‘mean of three independentplants’. Data were analysed statistically and standard errors werecalculated. Least significant differences (LSDs) between the meanvalues (n = 3) of control (WT and/or VC) and OsSUV3 overexpress-ing transgenic rice lines (L1–L3) were calculated by one-way analy-sis of variance (ANOVA) using SPSS 10.0 (SPSS, Inc., now IBM, http://www-01.ibm.com/software/analytics/spss). A comparison betweenthe means was performed using Duncan’s multiple range tests.The WT, VC and transgenic lines at P < 0.05, P < 0.01 andP < 0.001 were considered statistically significant.

Study of interactome of OsSuv3

The interactome of OsSuv3 was analysed using STRING 9.0 (http://string-db.org). The protein sequence of OsSUV3 was submittedand the results are presented in Figure S3.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the help of Drs Pawan Umateand Maryam Sarwat in the initial stages of the work, and MrDipesh Trivedi for Figure 1. We also thank Dr Sarvajeet Singh Gillfor his help in analysing the agronomical data. Work on plant heli-

cases and abiotic stress tolerance in N.T.’s laboratory is partiallysupported by the Department of Biotechnology (DBT), Govern-ment of India. We do not have any conflict of interest to declare.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. Comparison of amino acid sequences of Oryza sativa(Os) SUV3 (1–579) with SUV3 from Arabidopsis thaliana (At)(1–571), Homo sapiens (Hs) (1–786) and Saccharomyces cerevisiae(Sc) (1–737).Figure S2. Domain analysis of OsSUV3 and AtSUV3 proteins, andthe secondary structure of the OsSUV3 protein generated by thePDBsum server.Figure S3. Prediction of OsSUV3 protein-interacting proteins.

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OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice ( Oryza sativa L. cv. IR64)