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.
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
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).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
4 Narendra Tuteja et al.
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
20
40
60
80
100
120
WT VC L1 L2 L3
MD
A c
onte
nt (μ
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g–1
fw)
C 1h 6h 12h 24h
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lect
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WT VC L1 L2 L3
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ine
(μg
gm–1
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C 1h 6h 12h 24h(g)
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0102030405060708090
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% R
WC
C 1 h 6h 12h 24h(h)
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WT VC L1 L2 L3
CA
T u
nits
(mg
prot
ein)
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c
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APX
uni
ts (m
g pr
otei
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C 1h 6h 12h 24h(e)
c cc
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WT VC L1 L2 L3
GR
uni
ts (m
g pr
otei
n)–1
C 1h 6h 12h 24h(f)
aab
b ba ab
a abb
b
aab
b
ba b
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.
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
Rice SUV3 helicase functions in salinity tolerance 5
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).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
6 Narendra Tuteja et al.
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
0m
MNaC
l
Plantheight(cm)
78�
3.9a
34.66�
1.52
b80
�3.2a
75�
3.8a
78�
3.6a
74�
3.9a
83�
3.2a
79�
3.5a
Rootlength
(cm)
25�
0.97
a,b
11.66�
0.06
b28
�1.1a
24�
1.0a
,b29
�1.2a
24�
1.2a
,b31
�1.3a
27�
1.1a
Rootdry
weight(g)
2.88
�0.11
b0.92
3�
0.04
c4.1�
0.16
a3.5�
0.15
a4.0�
0.15
a3.5�
0.15
a4.8�
0.20
a3.9�
0.16
a
Leaf
area
(cm
2per
plant)
95�
2.7a
,b52
.83�
2.1c
110�
1.2a
97�
1.5a
,b11
2�
1.1a
96�
1.7a
,b10
9�
1.8a
97�
1.6a
,b
Totalch
lorophyll
(mgper
gfres
hweight)
9.48
�0.41
b2.05
�0.08
c9.77
�0.45
a9.67
�0.42
a9.82
�0.51
a9.79
�0.38
a9.95
�0.48
a9.83
�0.35
a
Totalprotein
(mgper
gfres
hweight)
19.18�
0.55
b8.01
4�
0.34
c26
.12�
0.88
a24
.15�
0.87
a,b
25.98�
0.91
a,b
24.25�
0.85
a,b
27.10�
0.85
a26
.71�
0.88
a
Net
photosy
nthetic
rate
(PN,lm
olCO
2m
�2s�
1)
10.45�
0.7b
6.93
�0.28
c12
.63�
0.68
a11
.23�
0.60
a12
.51�
0.71
a11
.37�
0.4a
12.07�
0.48
a11
.15�
0.5a
Stomatal
conductan
ce(gs,
mmolm
�2s�
1)
268�
15.4
a12
6.33
�5.9b
280�
13.89a
271�
16.5
a28
0�
13.94a
280�
11.8
a28
5�
15.32a
276�
11.4
a
Intrac
ellularCO
2
(Ci,lm
olmol�
1)
255�
15.2
a12
2.31
�4.7b
260�
14.52a
258�
11.5
a25
9�
13.96a
256�
11.4
a26
3�
12.54a
258�
10.5
a
Nitrogen
(%)
0.32
7�
0.01
1b0.10
7�
0.00
4c0.40
7�
0.01
2a0.41
5�
0.01
5a0.41
8�
0.01
4a0.42
7�
0.01
2a0.43
0�
0.01
3a0.43
1�
0.01
3a
Phosp
horus(%
)0.34
3�
0.01
0b0.12
23�
0.00
5c0.38
5�
0.01
1a0.38
2�
0.01
1a0.38
2�
0.01
2a0.38
1�
0.01
1a0.37
5�
0.01
2a0.37
3�
0.01
2a
Potassium
(%)
0.15
4�
0.00
4b0.07
4�
0.00
3c0.17
0�
0.00
4a0.16
8�
0.00
4a0.17
2�
0.00
3a0.16
6�
0.00
5a0.17
3�
0.00
4a0.16
8�
0.00
5a
Sodium
(%)
0.04
5�
0.00
1a0.06
3�
0.00
1a0.04
7�
0.00
1a0.04
7�
0.00
1a0.04
8�
0.00
1a0.04
8�
0.00
1a0.04
4�
0.00
1a0.04
4�
0.00
1a
Eac
hva
luereprese
nts
themea
nofthreereplic
ates
�SE.
Mea
nswereco
mpared
using
ANOVA.
Datafollo
wed
bythesa
melettersin
arow
arenotsignifica
ntlydifferentat
theleve
lofP>0.05
,as
determined
byaleas
t-significa
ntdifference
(LSD)test.a,b,cSignifica
ntdifference
sat
the
leve
lofP>0.05
,as
determined
byDunca
n’s
multiple
rangetest
(DMRT).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
Rice SUV3 helicase functions in salinity tolerance 7
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).
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
8 Narendra Tuteja et al.
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.
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
Rice SUV3 helicase functions in salinity tolerance 9
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.
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
10 Narendra Tuteja et al.
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
© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12277
Rice SUV3 helicase functions in salinity tolerance 11
(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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