Plant Physiology and Biochemistry 193 (2022) 1–13

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Enhanced osmotic adjustment, antioxidant defense, and photosynthesis

efficiency under drought and heat stress of transgenic cowpea
overexpressing an engineered DREB transcription factor
Sanjeev Kumar a, **, J. Muthuvel a, Ayan Sadhukhan b, Yuriko Kobayashi c, Hiroyuki Koyama c,
Lingaraj Sahoo a, *
a
Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, India
b
Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, 342030, India
c
Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, 501-1193, Gifu, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Cowpea is sensitive to drought and heat stress, particularly at the reproductive stages of development. Both
Cowpea stresses limit growth and yield, and their effect is more devastating when occurring concurrently. Dehydration-
Abiotic stress responsive element-binding protein 2A (DREB2A) is an important signaling hub integrating information about
Drought
two different abiotic stresses, drought and heat. We identified VuDREB2A as a canonical DREB ortholog in
Heat
cowpea, activating downstream stress-responsive genes by binding to DREs in their promoter. Post-translational
Transcription factor
Transcriptomics modification of a negative regulatory domain (NRD) within the VuDREB2A protein prevents its degradation.
Yield Targeted deletion of the NRD produces a stable and constitutively active form VuDREB2A-CA. However, there is
very little evidence of its practical utility under field conditions. This study overexpressed the VuDREB2A-CA in a
popular cowpea variety and conducted drought- and heat-tolerance experiments across various stress regimes.
Transgenic cowpea exhibited significant tolerance with consistently higher yield when exposed to over 30-
d drought stress and 3-d exposure to high temperature (28 ◦ C− 52 ◦ C) without any pleiotropic alterations. The
transgenic lines showed higher photosynthetic efficiency, osmotic adjustment, antioxidant defense, thermotol­
erance, and significantly higher survival and increased biomass than the wild type. Late embryogenesis abundant 5,
heat shock protein 70, dehydrin, mitogen-activated protein kinase 2/4, isoflavonoid reductase, and myoinositol phos­
phate synthase were upregulated in transgenic lines under drought and heat stress. Through transcriptome
analysis of the transgenic lines, we found significant up-regulation of various stress-responsive cowpea genes,
having DRE in their promoter. Our results suggest that overexpression of VuDREB2A could improve cowpea
production under drought and high temperatures.

1. Introduction physiological, and biochemical characteristics of the plants. An esti­

Cowpea [Vigna unguiculata (L.) Walp.] is an important legume
grown in semi-arid tropical regions of Africa, Asia, and America and
serves as a primary source of protein for millions of people. The supply
of protein with a high nutritional value and its ability of nitrogen fixa­
tion and adaptation to drought and high temperatures makes cowpea an
attractive legume for cultivation. Cowpea production faces numerous
constraints, of which abiotic stress, particularly drought, salinity, and
heat, critically affects productivity by altering several morphological,
mated cowpea production of more than 1000 kg grain ha− 1 is reduced
by 36–66% to 360 kg ha− 1 due to drought and heat stress, especially at
the flowering and pod filling stage (Powell, 2006; Bastos et al., 2011;
Nascimento et al., 2011).
Tolerances to drought and heat are complex quantitative traits
controlled by the cumulative effect of many genes or QTLs and are often
confounded by differences in the phenology of plants (Barnabas et al.,
2008; Fleury et al., 2010). Recent advances in genome mapping and
functional genomics technologies have provided powerful new tools for

* Corresponding author.
** Corresponding author.
E-mail addresses: sanjeev.bt@iitg.ac.in (S. Kumar), ls@iitg.ac.in (L. Sahoo).

https://doi.org/10.1016/j.plaphy.2022.09.028
Received 29 April 2022; Received in revised form 24 September 2022; Accepted 25 September 2022
Available online 15 October 2022
0981-9428/© 2022 Elsevier Masson SAS. All rights reserved.
S. Kumar et al.
the molecular dissection of drought tolerance (Worch et al., 2011). The
molecular markers and candidate genes were identified to understand
the molecular basis of drought tolerance better. Once the candidate
genes or traits associated with QTLs for drought tolerance are identified,
breeding with marker-assisted selection can be used to develop crops
with improved drought tolerance.
ABA-independent signaling pathways play essential roles in miti­
gating various abiotic stresses. As many as 1500 transcription factors
(TFs) interact with cis-elements in the promoter regions of different
stress-responsive genes to up-regulate the expression of many down­
stream genes to impart stress tolerance in Arabidopsis (Agarwal and Jha,
2010). DREB2 proteins are members of the
APETALA2/ethylene-responsive binding factor (AP2/ERF) family of
plant-specific TFs, which follow the ABA-independent signal trans­
duction pathway and recognize the core sequence motif A/GCCGAC,
termed the Dehydration-Responsive Element (DRE), in the promoters of
stress-inducible genes which code for osmoprotectants, reactive oxygen
species (ROS) scavengers and (Yamaguchi-Shinozaki and Shinozaki,
1994). The heat stress response of plants is usually achieved by heat
shock TFs, which induce the expression of heat shock proteins (HSPs).
HSPs are molecular chaperones that aid in proper protein folding and
homeostasis under heat stress. Hence, heat stress mitigation can be
achieved by engineering the HSP network (Fragkostefanakis et al.,
2015). Maintenance of plant growth and yield under drought and heat
stress critically depend on the sustenance of photosynthesis which
largely relies on the regulation of stomatal closure (Jia et al., 2021),
stability of chloroplast, and photosynthetic light-harvesting pigments
(Zhou et al., 2019) or the Rubisco enzyme under the harsh conditions
(Qu et al., 2021).
The DREB2A TF is an essential signaling hub in which two
completely different stresses, drought, and heat, converge (Liu et al.,
1998; Sakuma et al., 2006a,b). DREB2A orthologs are involved in
various plant species’ drought and heat tolerance mechanisms through
the transcriptional regulation of stress-responsive downstream genes
(Qin et al., 2007; Lata and Prasad, 2011; Mizoi et al., 2012; Chen et al.,
2016; Li et al., 2020). A negative regulatory domain (NRD) within
DREB2A destabilizes the protein by phosphorylation and
ubiquitin-mediated degradation (Mizoi et al., 2019). An essential
post-translational modification, i.e., SUMOylation in the DREB2A pro­
tein, prevents it from degradation under heat shock (Wang et al., 2020).
Earlier, we identified VuDREB2A from Vigna unguiculata, which showed
high induction under salt, dehydration, drought, and cold stress and
improved drought tolerance in transgenic Arabidopsis (Sadhukhan
et al., 2014). The drought tolerance of transgenic plants overexpressing
DREB genes has been tested mainly under greenhouse conditions.
However, recently (Zhou et al., 2019), overexpression of a DREB
ortholog from soybean in transgenic wheat increased the yield up to
18% under drought stress in the field by enhancing photosynthetic ef­
ficiency and production of osmoprotectants and preventing root damage
by increasing melatonin.
In the present study, we overexpressed a constitutively active form of
VuDREB2A in cowpea without the NRD, resulting in higher drought
adaptation. Improvement of cellular tolerance mechanisms in transgenic
cowpea overexpressing VuDREB2A-CA significantly improved the
intrinsic tolerance of cowpea, which increased the water and nutrient
uptake to sustain plant growth and development under drought stress.
We also observed significant up-regulation of drought-responsive genes
involved in signal transduction, MAPK2/4, heat shock proteins (HSPs),
and sugar metabolism (MIPS, IFR), leading to higher photosynthesis
efficiency and transpiration rate under both 30-d drought and 3-d termi­
nal heat stress. The transgenic cowpea plants significantly improved
canopy had better root/shoot growth and had higher productivity. Our
results demonstrate that introducing an engineered TF VuDREB2A can
be an effective strategy to engineer cowpea for better adaptability to
adverse environmental conditions, essential for sustainable agriculture
for future generations.
2
Plant Physiology and Biochemistry 193 (2022) 1–13
2. Materials and methods
2.1. Construction of binary vector and generation of cowpea
transformants
The modified cowpea DREB2A construct was prepared by deletion of
the negative regulatory domain between amino acid residues 132 and
182 to schedule a constitutively active (CA), putative active form
regulated through caulimovirus mosaic virus (CaMV) constitutive pro­
moter in an intermediate vector pRT101 (Sadhukhan et al., 2014).
Following digestion with HindIII, the 35S::VuDREB2A-CA:nos-Ter frag­
ment was inserted into binary plant vector pCAMBIA2301 that con­
tained kanamycin (nptII) as a plant selectable marker. Before the plant
transformation experiments, the binary construct was mobilized into
Agrobacterium tumefaciens strain EHA105 using triparental mating.
Cowpea transformants were generated from cotyledonary node explants
from a drought-sensitive variety cv. Pusa Komal, as previously described
(Kumar et al., 2017). The putative plants were hardened and subse­
quently transferred to soil in pots maintained to produce the homozy­
gous seeds in T3 and T4 generations. One-month-old putative
transformants were used for PCR screening and gene expression evalu­
ation. The confirmed transgenic lines were selected for drought and heat
stress treatments. Four randomly selected T0 PCR-positive events
(T0-#L3, T0-#L4, T0-#L5, and T0-#L7) were advanced up to T4 gener­
ations, and segregation studies were carried out to determine the in­
heritance of DREB2A, nptII, and gus by PCR.
2.2. Molecular characterization of transgenic cowpea lines
The cowpea genomic DNA from the putative plants was isolated from
young leaves using the DNASure Plant Mini Kit (Nucleopore, Genetix,
India). Presence of the transgenes nptII, gus, and 35S: DREB2A in
transformed plants (T0 –T4) was confirmed by PCR using gene-specific
primers. Stable integration of transgene and copy number was evalu­
ated by Southern hybridization by digesting 70 μg of genomic DNA each
from the WT and randomly selected independent T1 transgenic lines (T1-
#L3, T1-#L4, T1-#L5, T1-#L7 and T1-#L8) with EcoRI. The digested
genomic DNA was resolved on 0.8% agarose gel and blotted to a posi­
tively charged Zeta-Probe membrane (Bio-Rad, Hercules, CA, USA). The
blot was hybridized with the DIG-labeled 0.5 kb PCR product of the
coding region of nptII. The blot’s washing and detection were performed
according to the manufacturer’s instructions on the DIG labeling and
detection kit (Roche Diagnostics, Mannheim, Germany). For the relative
expression analysis of VuDREB2A, total RNA was extracted using the
RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), and cDNA was
synthesized using a cDNA synthesis kit (TAKARA, Japan) as per the
manufacturer’s instructions. The cDNA was used as a template to
perform RT-PCR analysis. Quantitative real-time PCR (qRT-PCR) was
performed with the fluorescent dye SYBR Green (QIAGEN) following the
manufacturer’s instructions. The relative expression levels of VuDREB2A
were calculated as described previously (Sadhukhan et al., 2014). The
stress-responsive genes HSP70, MAPK2/4, DHN1, LEA5, MIPS, and IFR
expression levels and the genes encoding antioxidant enzymes CAT,
APX, SOD, and POX were analyzed in cowpea exposed to 30-d drought
and 3-d terminal heat stress. The expression of the cowpea β-tubulin
gene served as an internal control.
2.3. Imposition of drought and heat stress in cowpea overexpressor lines
Drought and heat stress were imposed on 30-day-old mature control
(WT and Vector control) and transgenic plants grown in pots with 2.3 kg
soil. The pots were weighed daily to measure the water loss in evapo­
transpiration, and the required amount of water was poured to maintain
desired soil moisture status. At the 0-d of drought, all the assay pots were
covered with polybags to reduce osmo-transpiration. The stress expo­
sure continued till the desired soil moisture status. At the end of the
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

stress period, the plants were re-watered to 100% soil moisture status,
and after two weeks, recovery growth was recorded. Similarly, heat
stress was imposed on the same 30-d mature plants grown at 25 ◦ C under
70% relative humidity and 16 h/8 h [light/dark] photoperiod in a
growth chamber. For the heat stress experiments, the cowpea plants
were transferred to the growth chamber maintained at 28–45 ◦ C for 6 h
and at 45◦ C–52 ◦ C for 6 h and then returned to 28 ◦ C for 24 h recovery.
Experiments were carried out continuously for three consecutive days.

2.4. Transcriptome analysis of cowpea overexpressor lines and promoter

analysis of VuDREB2A-target genes

To perform an RNA-seq analysis of VuDREB2A-CA overexpressor

transgenic lines and WT cowpea, total RNA was isolated by Xcelgen
Plant RNA Kit. The RNA integrity and quality were estimated on 1.5%
formaldehyde denaturing agarose gel and quantified using a Nano­
drop8000 spectrophotometer. The RNA libraries were constructed using
the Illumina TruSeq mRNA library preparation kit. The library quality
and quantity were checked using Agilent DNA high sensitivity assay kit.
The paired-end sequencing was performed on the Illumina platform.
The promoter sequences up to 2 kb upstream to the transcriptional
start sites of the genes highly induced in the transgenic lines than the WT
were mined from the NCBI database (https://www.ncbi.nlm.nih.gov/).
These sequences were fed in the FASTA format to the New Plant Cis-
acting Regulatory DNA Elements (PLACE) web tool (https://www.dna.
affrc.go.jp/PLACE/?action=newplace) to identify the DRE cis-elements
in the promoters.

2.5. Analysis of photosynthetic activity under drought and heat stress

The photosynthetic activity was estimated based on the maximal

yield of the photochemical reaction in PSII (Fv/Fm). The Fv/Fm values
in the 3rd and 4th mature leaves from 1-month-old control and
VuDREB2A overexpressor lines were measured using a portable Chlo­
rophyll Fluorescence Meter (MINI-PAM-II, Photosynthesis Yield
analyzer (Heinz Walz GmbH, Germany) after 30 min of dark adaption.
Stomatal conductance and transpiration rates were measured by Leaf
Porometer (DECAGON Devices, USA). Measurements were made on
each plant in all four different transgenic lines with stressed and non-
stressed controls. The photosynthetic and chlorophyll fluorescence
measurements were taken between 10:00 a.m. and 12:00 p.m. when the
ambient light intensity was 1000–1200 μmol m− 2 s− 1.
2.6. Gas exchange measurements
Gas exchange analysis was carried out in 30-day-old mature and
control cowpea and the transgenic lines #L3, #L4, #L5, and #L7. The
control plant showed approximately − 0.201 MPa (no stress), − 1.20 MPa
(moderate stress), and − 1.72 MPa (severe stress) at predawn. All mea­
surements were done using LI-6800 Portable Photosynthesis System (LI-
COR, USA). Net photosynthesis rate (Pn), stomatal conductance (gs),
transpiration rate (E), and intracellular CO2 concentration (Ci) were
measured simultaneously between 11:00 a.m. and 1:00 p.m. under 1100
μmol m− 2 s− 1 artificial photosynthetic photon flux. The sample chamber
was programmed to maintain 400 ppm CO2 at 28 ◦ C and 50%–60%
relative air humidity for all measurements. The CO2 concentration in the
cuvette (Ca, μbar) was controlled using an 8 g CO2 cartridge in the
injector system. The plant leaves were acclimated in the leaf chamber for
2–4 min until the steady-state gas exchange was achieved. All stress and
control treatment measurements were conducted on the uppermost fully
expanded leaf in the third middle height of each plant. The leaves were
tagged to use the same sample in all measurements, and the mid-to-
distal- portions of each leaf blade were inserted into the LI-6800
chamber.
Fig. 1. Molecular characterization of VuDREB2A overexpression lines of
cowpea: (a) Schematic diagram of T-DNA region of the binary vector pCAM­
BIA2301-35SP::VuDREB2A; (b) PCR detection of 540 bp expected product with
nptII specific primer; (c) 460 bp expected product with 35SP-VuDREB2A spe­
cific primer and (d) 790 bp expected product with gus gene-specific primer;
Lanes M, 1 kb DNA marker; lane P, pCAMBIA23301-35SP∷VuDREB2A, positive
control; lane L2-L8, 35SP∷VuDREB2A transgenic lines; lane WT, untransformed
plant control; (e) Southern blot of EcoRI digested genomic DNA of 5 indepen­
dent T2 transgenic lines and WT, hybridized with nptII probe; (f) qRT-PCR based
expression analysis of VuDREB2A in four T2 transgenic cowpea lines and WT.
2.7. Physical and biochemical screening of cowpea overexpressor lines
under drought and heat stress
Leaves of control and transgenic cowpea lines T3-#L3, T3-#L4, T3-
#L5, and T3-#L7 were sampled before, post-drought, and heat stress
revival stage for the determination of relative water content (RWC),
MDA, ascorbate, and proline contents. RWC was determined as
described earlier (Lv et al., 2009). The leaves were floated on deionized
water for 5 h at 28 ◦ C, then the fresh weight was measured, after which
the hydrated leaves’ turgidity was recorded. Further, the leaves were
taken out, dried in a hot air oven at 80 ◦ C for 72 h, and weighed until a
constant dry weight (DW) was obtained. RWC was calculated using the
formula RWC (%) = (TW− DW)/(FW− DW) × 100.
Lipid peroxidation was estimated by measuring the MDA content

3
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Fig. 2. Response of VuDREB2A overexpression lines of cowpea to drought stress (a) 30-d old T3 transgenic and control before stress; (b) subjected to 30-d of drought
stress and; (c) 15-d post-recovery stage, bar = 10 cm. Control plants show chlorosis and wilting of leaves, whereas the transgenic plants show normal phenotype;
(d–e) Shoot (top) and root (bottom) phenotype analysis of different T3 transgenic and control plants at 15-d post-drought stress recovery, bar = 10 cm; (f) Com­
parison of root length and; (g) root/shoot growth ratio of control and different T3 transgenic lines grown on 30-d water deficit condition; Growth and yield
characteristics of cowpea transgenic lines and control; (h) agronomics yield of transgenic and control plants measured in terms of average pod length; (i) pod no./
plants; (j) seed no./plant (k) 100 seed weight/plant under >30d drought stress conditions. The data shows the mean ± S.E of three replicate samples. *Indicates
significant differences from the WT at P < 0.05. (T3-#L3, T3-#L4, T3-#L5, and T3-#L7), T3 transgenic lines; UWT; unstressed control, UT; unstressed transgenic, SWT;
stressed wild type, VC, Vector control.

described previously (Heath and Packer, 1968). Two hundred milli­
grams of fresh leaf tissue were homogenized with 2–3 ml of 0.25% TBA
containing 10% trichloroacetic acid. The obtained homogenate was
boiled in a water bath for 30 min at 95 ◦ C, cooled at room temperature,
and centrifuged at 10,000 g for 10 min. The absorbance was measured at
532 nm, and values corresponding to non-specific absorption at 600 nm
were subtracted. The lipid peroxidation rate equivalents were analyzed
according to the molar extinction coefficient of MDA, 155 mM− 1 cm− 1.
The MDA content was calculated using the equation: MDA (μmolg− 1FW)
= (6.45 × O.D.532) – (0.56 × O.D.450). Proline content was determined
by extracting the samples in 3% sulfosalicylic acid and measuring the
absorbance at 520 nm (Bates et al., 1973). The ascorbate content was
measured as described earlier (Panda et al., 2003).
2.8. Growth and yield analyses of cowpea overexpressor lines
Under normal, drought and heat stress conditions, the growth and
yield parameters were recorded at harvest time. Plant height, number of
branches, root and shoot lengths, root/shoot length ratios, biomass ra­
tios, and nodule formation in roots under normal and drought stress
conditions were measured. Simultaneously, the drooping of flowers and
formation of pods during and in post-3-d heat stress were recorded. The
yield was calculated in terms of the pod number, pod length, seed
number and the weight of 100 seeds.
3. Results
3.1. Development of transgenic cowpea lines with stably integrated
VuDREB2A-CA
The modified cowpea DREB2A construct was prepared by deletion of
negative regulatory domain between amino acid residues 132 and 182
to prepare a constitutively active (CA), putative active form regulated
through CaMV 35S constitutive promoter and with kanamycin selection
marker (Fig. 1a). Cotyledonary node explants of cowpea seedlings were
used to introduce 35S::VuDREB2A-CA using Agrobacterium-mediated
transformation (Kumar et al., 2017). Putative cowpea transformants
were initially identified in T0 and T1 generations by PCR screening fol­
lowed by Southern blotting. The amplified products, 540 bp nptII
(Figs. 1b), 460 bp DREB2A (Figs.. 1c), and 790 bp gus (Fig. 1d),
confirmed the presence of the T-DNA in kanamycin-resistant T0 trans­
genic cowpea. The segregation of the integrated genes DREB2A, nptII,
and gus in four T0 transgenic cowpea lines was followed up to T3 gen­
erations (T3-#L3, T3-#L4, T3-#L5, and T3-#L7) by the detection of
amplified products of expected sizes. Four of these three transgenic lines
(T4-#L3, T4-#L4, and T4-#L5) showed a 3:1 segregation ratio, having a
single T-DNA detected in these lines in Southern hybridization with a
0.540 kb nptII probe (Fig. 1e). The observed segregation ratios of
DREB2A, nptII, and gus revealed their inheritance in a Mendelian fashion

4
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Fig. 3. Response of VuDREB2A overexpression lines of cowpea to heat stress (a) Schematic map of heat stress imposition in transgenic cowpea lines; (b) 40-d old T3
transgenic and control before stress; (c) heat stress treatment (52 ◦ C) and after recovery at 28 ◦ C, Visible damage in the leaves of control plants after 6-hr heat stress
treatment and (d) 24-hr recovery, bar = 10 cm control plants show more significant damage in the form of wilting of leaves, whereas the transgenic plants show
normal phenotype; (e-f) shoot and root phenotype analysis of different T3 transgenic and control plants at 3-d post terminal heat stress recovery, bar = 10 cm; (g–h)
comparison of root length and root/shoot growth ratio of control and different T3 transgenic lines; (i) no of branches/plant before imposing heat stress; (j) no. of
flowers/plant before imposing heat stress; (k) no. of flowers formation in post 3-d heat stress; (l) no. of pod formation in post 3-d heat stress. The data shows the
mean ± S.E of three replicate samples. *Indicates significant differences from the WT at P < 0.05. (T3-#L3, T3-#L4, T3-#L5, and T3-#L7), T3 transgenic lines; WT,
wild-type cowpea; VC, vector control.

(Suppl. Table 1). The stable integration of T-DNA was observed in 4 out
of 5 lines in the T1 generation (T1-#L3, T1-#L4, T1-#L5, T1-#L7, and
T1-#L8) by genomic Southern blot analysis. Southern blot analysis of
transgenic lines showed that the T-DNAs were stably integrated into the
genome of transgenic cowpea lines in single and double copies. No hy­
bridization signal was observed in the WT (Fig. 1e). The DREB2A tran­
script levels of the four Southern-positive transgenic lines T1-#L3,
T1-#L4, T1-#L5, and T1-#L7 and WT cowpea were analyzed through
qRT-PCR after imposing 30-d drought stress. The lines showed >2-fold
expression compared to WT (Fig. 1f).
3.2. Overexpression of VuDREB2A in cowpea led to enhanced drought
tolerance
First, we examined the effects of drought stress on the growth of
control and T3 transgenic cowpea lines T3-#L3, T3-#L4, T3-#L5, and T3-
#L7 expressing VuDREB2A in mature plants under greenhouse condi­
tions. Under prolonged drought stress, the transgenic lines exhibited no
phenotypic difference between control and stress conditions. In contrast,
control plants under 30-d drought stress showed severe chlorosis, wilt­
ing, and growth inhibition (Fig. 2a–c). The control plants showed no
recovery after 15-d re-watering (Fig. 2c). However, the transgenic plants
remained green. They continued to grow normally, indicating that
transgenic plants were more tolerant to over 30 d of drought stress
(Fig. 2b). A significant increase in shoot biomass (Fig. 2d) and a 2.6–4.5-
fold more extended root system were observed in the transgenic cowpea
plants after 30 d of drought recovery at the 120-d stage (Fig. 2e and f).
The root/shoot growth ratio was 1.4− 3.9 fold higher in cowpea
overexpressing VuDREB2A-CA (Fig. 2g). These results indicated consti­
tutive expression of VuDREB2A led to drought-tolerant phenotypes
characterized by a significant increase in plant growth and performance
of transgenic plants under prolonged 30-d drought stress. Under stress
conditions we found that transgenic plants had bigger leaves, more leaf
area with improved yield such as average pod length (Fig. 2h), pod
number per plant (Fig. 2i), seed number per plant (Fig. 2j) and the
weight of 100 seeds per plant (Fig. 2k) in compare to control.
3.3. Overexpression of VuDREB2A-CA led to the enhanced tolerance of
cowpea to heat stress
We selected transgenic cowpea plants for terminal heat stress during
the reproductive stage. We presented the schematic map of heat stress
imposition in control and transgenic cowpea lines (Fig. 3a). We found
that cowpea VuDREB2A overexpressing lines T3-#L3, T3-#L4, T3-#L5,
and T3-#L7 showed enhanced tolerance to 3-d heat stress (ranging from
28 ◦ C − 45 ◦ C for 6 h, and 45 ◦ C − 52 ◦ C for 6 h) in comparison to control
plants (Fig. 3b–d). Under normal conditions (28 ◦ C), the phenotypes of
transgenic cowpea lines did not differ from the control plants in terms of
plant growth and flowering. However, when we treated 40-old control
and transgenic cowpea plants with heat stress (45 ◦ C − 52 ◦ C) for 6 h, the
transgenic plants exhibited marked thermotolerance compared to con­
trol plants (Fig. 3c). The plants were kept for recovery by gradually
decreasing the temperature from 52 ◦ C to 28 ◦ C, held for 24 h; severe
damage was observed in control plants in the form of wilting and flower
droop, whereas the transgenic plants exhibited only slight wilting with
intact flower (Fig. 3d). We also investigated the plant growth parameter

5
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Fig. 4. Measurement of photosynthetic efficiency of VuDREB2A overexpression lines of cowpea under 30-d drought and 3-d heat stress (a) PSII photosynthetic
efficiency (Fv/Fm) in the leaves of control and transgenic cowpea plants under 30-d drought stress; (b) Fv/Fm in the leaves of WT and transgenic plants after 3-d heat
stress treatment; (c) Transpiration rate under 30-d drought stress; (d) Transpiration rate under 3-d heat stress; (e) Stomatal conductance under 30-d drought stress;
(f) Stomatal conductance under 3-d heat stress. The data shows the mean ± S.E of three replicate samples. *Indicates significant differences from the WT at P < 0.05.
(T3-#L3, T3-#L4, T3-#L5, and T3-#L7), T3 transgenic lines; WT, wild-type cowpea; VC, vector control.

such as root and shoot growth in post-recovery of both transgenic and
control (Fig. 3e–h). We found significant variation in the shoot (Fig. 3e)
and root growth (Fig. 3f) in comparison to control plants. We observed
the plant growth in terms of no. of branches formed (Fig. 3i) and no. of
flowers developed (Fig. 3j) before inducing heat stress. Pod formation in
post heat stress recovery stage was severely affected in control plants;
among a total of 8 flowers, only two flowers were able to form pods,
while in transgenic lines, among ten flowers, all formed uniform pods
with a good number of seeds (Fig. 3k–l). These results implicated that
cowpea lines overexpressing VuDREB2A-CA triggered plant growth
performance during the reproductive stage.
3.4. Overexpression of VuDREB2A-CA led to an improved photosynthetic
yield of cowpea under drought and heat stress
To investigate the changes in PSII level, photoinhibition, utilization,
and dissipation of excess excitation energy in drought and heat-stressed
cowpea overexpressor lines, measurements of various fluorescence pa­
rameters related to PSII photochemistry were analyzed under stressed
and non-stressed conditions. We found a 1.8–2.3-fold difference in the
Fv/Fm ratio between the control and transgenic cowpea (Fig. 4a). Under
the heat stress condition, we observed the Fv/Fm of control plants
decreased by 1.1–1.5-fold as compared to transgenic lines T3-#5 and T3-
#7. After 24 h recovery, transgenic cowpea lines’ photosynthetic effi­
ciency returned to normal. In contrast, control plants continued to show
reduced photosynthetic efficiency (Fig. 4b). The transpiration rate at the
initial stage of drought (0–10 d), the transpiration rate was identical to
the control value, but at the progressive stage (10–30 days), the tran­
spiration rate was found to be significantly higher from 0.82 to 1.2-fold
(Fig. 4c) while the rate was little lesser from 0.8 to 1.1-fold under heat
stress (Fig. 4d). Stomatal conductivity was found 0.9–1.1-fold higher in
transgenic cowpea plants compared to the control plants in both drought
as well as heat stress (Fig. 4e–f). At the beginning of water withdrawal,
cowpea plants’ leaf water potential (LWP) was always at − 0.5 MPa or
greater. After 30 d of drought treatment, LWPpred of the progeny of the
transgenic line T3-#7 and line T3-#4 reached − 1.72 MPa and − 1.50
MPa, respectively, while the LWPpred of the control plants was − 1.68
MPa (Supplementary Fig. 1).
Concerning LWPpred thresholds for gas exchange analysis, the leaf
water status was allowed to progress until approximately − 1.20 MPa
(moderate stress) and − 1.72 MPa (severe stress) in control plants,
considering the more accelerated decline in LWPpred values in control
compared to the transgenic lines, as described earlier (Lisei-de-Sá et al.,
2017). Increased water deficit led to greater gas exchange performance
in transgenic cowpea plants compared with control under drought and
heat stress (Fig. 5). The Pn values were lower in control while
1.5− 2.5-fold higher in transgenic lines. We observed the highest 2.5-
fold in Line T3-#7 (Fig. 5a–b), which indicated that cowpea lines over­
expressing VuDREB2A improved their net photosynthetic rate even
when maintained under water deficit conditions.
Gs values were influenced by drought and heat stress, but lines T3-#
4 and T3-#7 showed the highest 2- and a 3-fold increase compared to the
control under drought and drought heat stress, respectively (Fig. 5c–d).
A similar increase in transpiration rate (E) ranging from 1.7 to 2.2- fold
in transgenic lines was observed both under drought and heat stress
(Fig. 5e–f). We also observed an increase in Ci in all transgenic lines
compared to control under drought and heat stress (Fig. 5g–h). As the
drought stress treatment progressed, the proportional decrease in Pn was

6
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Fig. 5. Gas Exchange Measurements of VuDREB2A overexpression lines of cowpea under 30-d drought and 3-d heat stress (a) Net photosynthetic rate Pn; under 30-
d drought stress; (b) Net photosynthetic rate Pn; under 3-d heat stress (c) Stomatal conductance gs; under 30-d drought stress; (d) Stomatal conductance gs; under 3-
d heat stress; (e) Transpiration rate E under 30-d drought stress; (f) Transpiration rate E under 3-d heat stress; (g) Intercellular carbon concentration Ci under 30-
d drought stress; (h) Intercellular carbon concentration Ci under 3-d heat stress monitored in control and transgenic lines. Drought stress treatments were carried out
according to leaf water status: − 0.201 MPa (no stress) and − 1.72 MPa (severe stress) at predawn. Each bar represents the average of three replicates ± standard
error. Statistical analysis was performed according to the Student’s t-test (p < 0.05). The data shows the mean ± S.E of 3 independent replicates. *Indicates sig­
nificant differences from the WT at P < 0.05. (T3-#L3, T3-#L4, T3-#L5, and T3-#L7), T3 transgenic lines; WT, wild-type cowpea; VC, vector control.

significantly less affected than the decrease in stomatal conductance, compared to the transgenic lines. A similar decreasing trend in the value
demonstrating the different roles of stomatal responses on the gas ex­ of E and Ci was observed in control plants under drought and heat stress.
change parameters in the analyzed lines, i.e., lines T3-#3, T3-#4, T3-#5 These results suggest that overexpression of VuDREB2A in cowpea
and T3-#7. There was a sharp decrease in gs in the control plants improved the photosynthetic yield under drought and heat stress

7
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Fig. 6. Drought and heat stress response of VuDREB2A overexpression lines of cowpea. (a) Relative water content (percentage) in leaves of control and transformed
T3 lines exposed to 30-d drought stress; (b) Relative water content (%) in leaves of control and transformed T3 lines exposed to 3-d terminal heat stress; (c) Changes in
the level of proline accumulation under 30-d drought stress; (d) Changes in the level of proline accumulation under 3-d terminal heat stress. The data shows the mean
± S.E of three replicate samples. *Indicates significant differences from the WT at P < 0.05. (T3-#L3, T3-#L4, T3-#L5, and T3-#L7), T3 transgenic lines; WT, wild-type
cowpea; VC, vector control.

conditions. d), MDA content significantly increased in the leaves of control plants. In
contrast, the transgenic lines T3-#L3, T3-#L4, T3-#L5, and T3-#L7
demonstrated a sharp 2.1–3.0-fold decrease in MDA content under
3.5. Overexpression of VuDREB2A-CA led to the enhanced production of
drought (Fig. 7a) and 1.3–2.1-fold decrease under heat stress (Fig. 7b).
osmoprotectants and ROS scavenging
The lower MDA content in the transgenic lines indicated a lower
membrane injury under drought and heat stress than the control. We
Plant survival under water and heat stress conditions depends on
observed 1.9–2.7-fold higher ascorbate content in the transgenic cowpea
maintaining water balance inside the cell. Therefore, we examined the
lines under 30-d drought (Figs. 7c) and 1.3–2.3-fold higher under 3-
leaf RWCs in all four cowpea transgenic lines, T3-#L3, T3-#L4, T3-#L5,
d heat stress than in control (Fig. 7d). The increase in ascorbate con­
T3-#L7 and control plants during 30-d drought and 3-d heat stress. After
tent may help to mitigate oxidative damage in the DREB2A over­
the imposition of prolonged 30-d drought stress, leaf RWC declined in
expressing cowpea lines.
control plants by 1.5-fold, unaffected in the transgenic lines (Fig. 6a).
We performed H2O2 and O−2 detection using DAB and NBT staining to
The transgenic lines demonstrated approximately 1.2–1.6-fold higher
study whether VuDREB2A overexpression affects the redox balance. A
potential to maintain water in leaf tissues than control under drought
Brown precipitate formation was detected in the control and transgenic
stress (Fig. 6a). Similarly, RWC was found 0.7–1.2-fold higher in leaves
lines under drought and heat stress indicating polymerization of DAB by
under 3-d heat stress in the transgenic lines than in control (Fig. 6b).
H2O2. DAB staining was stronger in control leaves compared with
Osmolytes such as proline protect the cell against various abiotic
transgenic lines after 30-d drought and 3-d heat stress treatment,
stresses such as drought, salinity, and heat. To evaluate the possible role
whereas the transgenic lines T3-#L3, T3-#L4, T3-#L5, and T3-#L7
of VuDREB2A overexpression in cowpea under drought and heat stress,
showed significant tolerance to H2O−2 induced damage. We found higher
proline contents were measured in control, and all four transgenic
tolerance in line #3 and line #4 compared to line #5 and line #7 under
cowpea lines T3-#L3, T3-#L4, T3-#L5, and T3-#L7. No significant dif­
drought stress. However, lines #3, #5, and #7 showed better tolerance
ference in the proline content was observed between the control and
than line #4 under heat stress (Fig. 7e–f). Through NBT staining, we
transgenic lines without stress. In contrast, after 30-d drought exposure,
detected a lower accumulation of O−2 in the form of white stain in the
the transgenic plants recorded a significantly 3.4–4.8-fold higher proline
transgenic cowpea lines, while intense blue color was observed in con­
accumulation than control plants (Fig. 6c). Similarly, proline accumu­
trol. We found better tolerance in transgenic line #3 and line #4
lation was 1.8–3.3-fold higher after 3-d heat stress exposure in the
compared to line #5 and line #7 under 30-d drought stress. In contrast,
transgenic lines in comparison to control (Fig. 6d). Our results indicated
line #4 and line #5 were better than line #3 and line #7 under 3-d heat
that constitutive overexpression of VuDREB2A led to higher RWC and
stress (Fig. 7f), indicating lower O−2 generation and lesser oxidative
proline contents in cowpea under both drought and heat stress.
damage in VuDREB2A overexpressor lines after drought and heat stress
Drought and heat stress exerts oxidative and osmotic stress in plant
exposure. These results showed that under drought and heat stress, the
cells. ROS-scavenging is a well-known phenomenon that plays a vital
transgenic plants exhibited considerably less accumulation of H2O2 and
role in the tolerance of plants to various abiotic stresses. Therefore, we
superoxide radicals relative to control.
determined the lipid hydroperoxide production rate changes by deter­
mining malondialdehyde (MDA) and ascorbate content in the control
leaves and transgenic cowpea overexpressing VuDREB2A under drought
and heat stress. At the prolonged stage of deficiency (30- d) and heat (3-

8
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Fig. 7. ROS mediated antioxidant metabolite response of VuDREB2A overexpression lines of cowpea under drought and heat stress (a) Levels of lipid peroxidation
expressed in terms of MDA content under 30-d drought stress; (b) MDA content under 3-d terminal heat stress; (c) Ascorbate content under 30-d drought stress; (d)
Ascorbate content under 3-d terminal heat stress in the leaves of control and T3 transgenic lines; (e) Histochemical analysis of oxygen radicals and hydrogen peroxide
in leaves of control and transformed T3 transgenic lines exposed to 30-d drought stress; (f) and at 3-d terminal heat stress. The data shows the mean ± S.E of three
replicate samples. *Indicates significant differences from the WT at P < 0.05. (T3-#L3, T3-#L4, T3-#L5, and T3-#L7), T3 transgenic lines; WT, wild-type cowpea; VC,
vector control.

3.6. Overexpression of VuDREB2A-CA led to induced expression of stress-
responsive genes under drought and heat stresses
To identify the genes that are upregulated by the overexpression of
VuDREB2A, we performed expression profiling in control and transgenic
lines T3-#L3, T3-#L4, T3-#L5, and T3-#L7 under both drought and heat
stress (Fig. 8a-l). We observed 1.2–1.5-fold increase in the expression of
myoinositol phosphate synthase (MIPS) (Fig. 8a–g), 1.9–2.5-fold in late
embryogenesis abundant group-5 (LEA-5) (Fig. 8b–h), 2.5–3.0-fold in
dehydrin (DHN1) (Fig. 8c–i), 1.8–2.5-fold mitogen-activated protein ki­
nase-2/4 (MAPK2/4) (Fig. 8d–j), 2.3–2.7-fold in heat shock protein 70
(HSP70) (Fig. 8e–k) and 0.6–0.9-fold in isoflavonoid reductase (IFR)
(Fig. 8f and l) in the transgenic lines than the control under 30-d drought
and heat stress (33-52 ◦ C). Simultaneously we also checked the
expression level of ROS-induced antioxidative enzyme genes (Fig. 9a–h)
such as catalase (CAT) (Fig. 9a & e), superoxide dismutase (SOD) (Fig. 9b
& f), ascorbate peroxidase (APX) (Fig. 9c & g), and peroxidase (POX)
(Fig. 9d & h). We observed 0.8–3.0-fold higher expression under 30-
d drought and 1.2–2.9-fold under 3-d heat stress, respectively, in all
transgenic lines (Fig. 9a–h).
3.7. Identification of VuDREB2A-target genes under drought stress
through transcriptome analysis
We performed RNAseq analysis of two highly expressing transgenic
lines T3-#5 and T3-#7, along with control cowpea after 30-d drought
stress treatment. The genes up-regulated in 35S: VuDREB2A-CA lines
concerning the control were extracted (Suppl. Table 2). Among them,
the 20 most highly induced genes in both the transgenic lines (log2fold-
change > 5, false discovery rate <0.05) containing DRE in their pro­
moters are presented in Table 1. Detection of the DRE cis-elements in the
promoter of these genes suggested their direct regulation by VuDREB2A
in cowpea. These genes included several TFs, including ERF027-like,
MYB48-like, MYB41-like, MYB13-like, MYB86-like, bHLH94-like, GLA­
BRA 3-like isoform X2, GATA 22, and TGA2.3-like, ion transporters like
mechanosensitive ion channel protein 6-like, cation/H(+) antiporter 19-
like, KAT8, and ABC transporter G family member 11-like, cell wall and
Casparian strip-related proteins like CASP-like protein 1C1, a soyasa­
ponin biosynthesis gene, soyasapogenol B glucuronide
galactosyltransferase-like, a melatonin biosynthesis gene, acetylser­
otonin O-methyltransferase-like, and an ethylene biosynthesis gene,
putative 1-aminocyclopropane-1-carboxylate synthase 3-like (Table 1).
The induced expression level of these genes in the overexpressor lines
#L5, #L7, and WT were cross-validated by qRT-PCR. The qRT-PCR
expression analysis revealed that the relative expression trends of 6
putative target genes in response to the drought stress treatment were
the same as that was predicted by transcriptome sequencing (Suppl.
Fig. 2).
3.8. Overexpression of VuDREB2A-CA led to increased grain yield under
both drought and heat stress
Cowpea plants overexpressing VuDREB2A subjected to 30-d drought
and 3-d heat stress were also analyzed for phenotypic characteristics

9
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Fig. 8. Relative expression levels of VuDREB2A-regulated stress-responsive genes under drought and heat stress in transgenic cowpea lines (a) real-time expression
levels of myoinositol phosphate synthase (MIPS); (b) late embryogenesis abundant-5 (LEA-5); (c) dehydrin 1 (DHN1); (d) mitogen-activated protein kinase (MAPK2/4); (e)
heat shock protein 70 (HSP70) and; (f) isoflavonoid reductase (IFR) under 30-d drought stress; (g) real-time expression levels of MIPS; (h) LEA-5; (i) DHN1; (j) MAPK2/
4; (k) HSP70 and; (l) IFR under 3-d heat stress in control and four T3 transgenic cowpea lines; Vu-β tubulin gene served as an internal control. UWT; unstressed
control, UT; unstressed transgenic, SWT; stressed control, Line T3-#L3, T3-#4, T3-#5, T3-#7 are stressed transgenic lines; VC, vector control.

Fig. 9. Relative expression levels of ROS-mediated antioxidant enzymatic genes under drought and heat stress in transgenic cowpea lines (a) real-time expression
levels of stress-responsive genes under 30-d drought stress, catalase (CAT); (b) superoxide dismutase (SOD); (c) ascorbate peroxidase (APX) and; (d) peroxidase (POX);
(e) real-time expression levels of stress-responsive genes under 3-d heat stress of CAT; (f) SOD; (g) APX and; (h) POX in control and four T3 transgenic cowpea lines;
Cowpea β-tubulin gene was used as an internal control, UWT; unstressed control, UT; unstressed transgenic, SWT; stressed control, Line T3-#L3, T3-#4, T3-#5, T3-#7
are stressed transgenic lines; VC, vector control.

such as plant height, shoot biomass, root biomass, root/shoot ratio,
average number of pods per plant, average pod length, number of pods
per plant, and weight of 100 seeds per plant. Under drought and heat
stress, we observed that transgenic plants had bigger leaves, more leaf
area, 3–4.5-fold increased root length, and a 1.8–2-fold higher root/
shoot ratio than the control plants (Figs. 2 and 3). All four transgenic
lines stayed green and started producing new leaves, flowers, and pods
even after 120 days. In contrast, control plants showed yellowish leaves
with senescence symptoms only after 2nd week of drought stress
(Fig. 2b). No significant yield loss was observed in the overexpressor
lines under drought stress compared to normal unstressed WT and
vector control plants. The transgenic line T3-#5 had the highest number
of pods/plant than stressed and unstressed WT plants under greenhouse
conditions, while lines T3-# 3, T3-#4, and T3-#7 plants had an

10
S. Kumar et al.
approximately similar number of pods/plant compared to unstressed
control plants. The plant biomass was higher in all four overexpressor
lines than control, which showed positive correlations with pod yield
and seed weight, indicating that the increased yield was due to growth
enhancement in the transgenic lines.
4. Discussion
Drought and heat stress detrimentally affect the growth and pro­
ductivity of plants. The experience of drought stress by plants under
rain-fed conditions is complex as its magnitude and duration are un­
predictable and affected by soil texture and vapor pressure deficit. Plants
have evolved several adaptive mechanisms to cope with diverse drought
stress situations, including reducing the transpiration rate, conserving
water, and lengthening the roots inside the soil to maintain water supply
(Blum, 2005). Besides, when the tissue water potential decreases under
prolonged drought stress, the tolerance mechanisms at the cellular level
assume significance in sustaining metabolic activities (Granier et al.,
2006). Plant traits associated with water relations and diverse cellular
tolerance mechanisms are crucial to achieving field-level drought
tolerance. Identifying stress-specific TFs is an essential prerequisite for
crop improvement. Among the various TFs, DREB is one of the most
promising candidate genes conferring abiotic stress tolerance in several
crops (Kizis et al., 2001; Roorkiwal et al., 2014). Ectopic expression of
DREB genes in Arabidopsis (Cong et al., 2008; Saleh et al., 2006), as well
as in heterologous systems such as wheat, barley, soybean, tomato, to­
bacco, strawberry, rice, oilseed rape, potato, and other grasses, have also
shown enhanced tolerance to one or more types of abiotic stresses (Cong
et al., 2008; Behnam et al., 2007; Chen et al., 2007, 2008; Cong et al.,
2008; Ito et al., 2006; Morran et al., 2011; Oh et al., 2007; Wang et al.,
2008; Zhao et al., 2007). Earlier, we reported a novel VuDREB2A from
cowpea as a canonical DREB2-type TF, which can bind DRE in vitro and
confer enhanced drought resistance in transgenic Arabidopsis (Sadhu­
khan et al., 2014). A constitutively active form of VuDREB2A without a
negative regulatory domain rendered the transgenic plant tolerant to
drought and salt stresses and heat, which indicates a role for DREB2A in
crosstalk between drought and heat-stress responses. An earlier report
demonstrated that DREB2A controls the expression of the heat stress TF
HsfA3 (Schramm et al., 2008). Similarly, overexpression of ZmDREB2A
in maize under constitutive or stress-inducible promoter resulted in
enhanced drought tolerance in transgenic plants (Qin et al., 2007). This
study successfully overexpressed VuDREB2A-CA in the drought-sensitive
variety Pusa Komal. The transgenic cowpea lines, subjected to gradual
drought and heat stress, showed significantly improved tolerance and
higher grain yield than the control. Under the stress conditions, all
transgenic cowpea lines grew well, flowered normally, and set uniform
pods and viable seeds even under 30-d drought stress. At the same time,
control plants showed severe chlorosis and withered with a significant
reduction in yield-related traits. However, no significant difference was
observed in plant yield in unstressed cowpea plants. The transgenic
cowpea lines showed optimal vegetative growth and a significantly
better survival ratio than control under drought (Fig. 2h–k) and heat
stress (Fig. 2j–k) conditions.
Roots are an essential plant organ absorbing mineral nutrients and
soil moisture. With a deep and thick root system, plants can gain better
access to water and show higher drought tolerance (Jeon et al., 2011).
Previously, (O’toole and Bland, 1987) reported that rice varieties with
thicker roots were more tolerant to drought than those with thinner
roots. The transgenic cowpea lines expressing VuDREB2A showed
3.0–4.5-fold longer roots with more root nodules under 30-d drought
stress conditions (Fig. 2d). Our findings and results indicate that thick­
ened roots with increased shoot biomass are majorly responsible for
increased tolerance, leading to increased grain yield under drought
stress conditions.
Prolonged drought and heat stress cause a cellular water imbalance,
inducing ROS generation and consequently triggering the degradation of
11
Plant Physiology and Biochemistry 193 (2022) 1–13
chlorophyll and membrane lipid peroxidation. The adverse effect of
drought stress directly affects photosynthesis efficiency, stomatal con­
ductivity, and transpiration rate, reducing overall plant growth and
yield. In our study, the transgenic cowpea lines overexpressing
VuDREB2A were found to maintain higher chlorophyll fluorescence ra­
tios (Fv/Fm), higher transpiration rates, and higher stomatal conduc­
tivities under 30-d drought and 3-d heat stress in comparison to the
control plants (Fig. 3a), which indicates better photosynthetic efficiency
under prolonged drought and terminal heat stress. Transgenic cowpea
overexpressing VuDREB2A lines (T3-#L3, T3-#L4, T3-#L5, and T3-#L7)
showed lesser damage to the photosynthetic apparatus, maintaining the
normal growth and yield. At the same time, the control plants experi­
enced severe impairment of their photosynthesis machinery under 30-
d drought and 3-d heat stress (Fig. 4). The observed results indicate
that VuDREB2A increased the protection of the photosystem in the
transgenic plants to lessen the effects of drought and heat stress on
photosynthesis.
In this study, we observed that all the gas exchange variables were
higher in the transgenic lines compared to control plants as drought and
heat stress intensity progressed from − 1.20 MPa (moderate stress) to
− 1.72 MPa (severe stress) (Fig. 5). This observation reflected the more
extraordinary ability of the transgenic plants to acquire a more signifi­
cant amount of water because of their deeper and increased root biomass
(Lisei-de-Sá et al., 2017). These results also suggest that these transgenic
lines could tolerate a sub-optimal water supply, most likely due to the
situational activation of the stress-responsive VuDREB2A gene driven by
the constitutive 35S promoter.
Transgenic rice overexpressing OsDREB1A demonstrated improved
tolerance to drought, high salt, and low-temperature stresses, and
accumulated elevated levels of osmoprotectants such as free proline and
soluble sugars (Ito et al., 2006). Our study observed high levels of
endogenous proline, leaf RWC (Fig. 6), and other soluble sugars in
VuDREB2A overexpressing lines under 30-d drought and 3-d heat stress
but not under non-stressful conditions, which indicates possible
involvement in cellular protection. During drought stress, the cellular
osmotic potential is restored by accumulating proline inside the cell,
which functions as a signaling molecule that can even regulate gene
expression essential for stress mitigation (Szabados and Savoure, 2010)
(Fig. 6).
Drought and high-temperature stress induced a noticeable accumu­
lation of ROS in the plants, which poses a significant threat to crop
productivity (Boyer, 1982). ROS, including superoxide (O.-2 ) and
hydrogen peroxide (H2O2), are synthesized in organelles such as chlo­
roplasts, mitochondria, and peroxisomes. However, it is getting highly
stimulated under various abiotic stressed conditions. Under prolonged
drought and heat stress, this balance suffers an upward shift, ROS pro­
duction being enhanced due to stomatal closure and the concomitant
limitation on CO2 fixation (Boo and Jung, 1999). ROS produced by these
stresses is toxic molecules capable of causing oxidative damage to
cellular proteins, cell membranes, nucleic acids, lipids, and carbohy­
drates (Foyer and Noctor, 2005). The ROS component, such as H2O2,
also plays an essential role in signaling molecules that trigger defense
responses to various biotic and abiotic stresses. In contrast, it stimulates
programmed cell death (Quan et al., 2008). In the current study, while
H2O2 and O.-2 levels significantly increased in control cowpea plants
under drought and heat stress, these levels were found significantly
lower in all four transgenic lines expressing VuDREB2A compared to
control plants under these conditions. In addition, VuDREB2A over­
expression lines showed significantly less lipid peroxidation (Fig. 7),
increased level of ascorbate content, and expression of antioxidant
enzyme genes like CAT, SOD, APX, and GR under prolonged drought and
terminal heat stress conditions (Fig. 9). These 4, CAT, SOD, APX, and
POX, are the major enzymes responsible for H2O2 scavenging during
oxidative stress in plants. Besides H2O2 scavenging, plant APXs are also
involved in plant growth and development and the lignification and
cross-linking of cell wall components (Passardi et al., 2005). These data
S. Kumar et al. Plant Physiology and Biochemistry 193 (2022) 1–13

Table 1 highly expressing transgenic cowpea lines, T3-#L3 and T3-#L7. Through
Putative target genes of VuDREB2A in cowpea. transcriptome analysis of transgenic cowpea, we found various putative
S/ Gene identifier Log2(fold P- Gene Description direct target genes of VuDREB2A in cowpea, including many transcrip­
N change) Value tion factors, different ion transporter genes, enzymes involved in cell
1 XM_028082815.1 7.92 2.4E- acetylserotonin O- wall modification and ethylene biosynthesis, genes involved in the
11 methyltransferase-like biosynthesis of the protective phytochemicals melatonin and soyasa­
2 XM_028049101.1 7.60 1.4E- putative GATA transcription ponin, sugar transporter, and photosynthetic genes including cyto­
09 factor 22 chromes, whose products synergistically led to the alleviation of drought
3 XM_028057542.1 7.26 5.6E- 1-aminocyclopropane-1-
08 carboxylate synthase 3-like
stress in cowpea (Table-1). Further analysis of the function of these
4 XM_028055578.1 7.18 1.2E- CASP-like protein 1C1 genes by reverse genetics will throw more light on the drought stress
07 signaling in cowpea through VuDREB2A. Finally, our results confirm
5 XM_028056020.1 7.10 2.6E- cation/H(+) antiporter 19- that the overexpression of engineered TF genes like VuDREB2A is a
07 like
potential approach to improving cowpea production under abiotic
6 XM_028047992.1 7.01 5.9E- transcription factor TGA2.3-
07 like stress. This study will provide a firm foundation for improving drought
7 XM_028079556.1 6.92 1.3E- sugar transport protein 13- and heat stress tolerance in other legumes and cereal crops.
06 like
8 XM_028064766.1 6.81 1.1E- (3S,6E)-nerolidol synthase 1-
Author contributions
31 like
9 XM_028071067.1 6.76 2.3E- uncharacterized isomerase
45 BH0283-like L.S, H.K, Y.K, and S.K: Conceptualization. L.S, A.S, and H.K: Data
10 XM_028059453.1 6.72 7.0E- soyasapogenol B glucuronide curation. S.K, M.J, A.S: Formal analysis. S.K, M.J, and L.S: Investigation.
06 galactosyltransferase-like L.S: Funding acquisition. S.K, M.J, and L.S: Methodology. L.S, Y.K, and
11 XM_028062906.1 6.72 7.0E- ABC transporter G family
H.K: Project administration. L.S: Resources. S.K and L.S: Writing –
06 member 11-like
12 XM_028081344.1 6.60 1.6E- mechanosensitive ion channel original draft. A.S: Writing – review & editing. All authors have read and
05 protein 6-like agreed to the manuscript.
13 XM_028052834.1 6.34 9.5E- ethylene-responsive
05 transcription factor ERF027-
like Declaration of competing interest
14 XM_028070267.1 6.03 1.8E- transcription factor MYB48-
17 like The authors declare that they have no known competing financial
15 XM_028053534.1 6.03 5.9E- transcription factor MYB41-
interests or personal relationships that could have appeared to influence
04 like
16 XM_028066213.1 6.01 6.1E- transcription factor MYB13- the work reported in this paper.
27 like
17 XM_028046621.1 5.84 1.5E- transcription factor bHLH94- Data availability
03 like
18 XM_028064829.1 5.62 4.0E- transcription factor GLABRA
03 3-like isoform X2
The authors are unable or have chosen not to specify which data has
19 XM_028061056.1 5.62 4.0E- KAT8 regulatory NSL complex been used.
03 subunit 3
20 XM_028046616.1 5.36 1.1E- transcription factor MYB86- Acknowledgments
02 like

Genes upregulated in two 35S::VuDREB2A-CA lines are shown (log2fold change
>5; false discovery rate < 0.05). Fold change refers to the ratio of the expression
levels in 35S::VuDREB2A-CA to that in the WT. The t-test P-values are shown.
Genes were annotated from the NCBI Entrez database. The dehydration
responsive elements (DREs) were detected in the promoter sequences up to 2 kb
upstream to the transcription start site of each gene using the New Plant Cis
Acting Regulatory DNA elements (PLACE) database.
suggest that overexpression of VuDREB2A might play an essential role in
regulating drought and heat tolerance by controlling the antioxidant
machinery.
VuDREB2A-CA overexpression also resulted in 2.4–5.6-fold upregu­
lation of other stress-responsive genes such as HSP70, DHN1, and
osmoprotectant genes like LEA-5, signaling genes like MAPK2/4. Sugar
synthesis genes such as MIPS and IFR on exposure to 30-d drought and 3-
d heat stress (Fig. 8). This corroborated with earlier reports where LEA
group genes were found several folds upregulated under drought stress
(Magwanga et al., 2018), increased IFR expression was observed in
soybean under water deficit (Gutierrez-Gonzalez et al., 2010), a
myo-inositol-1-phosphate synthase gene, IbMIPS1 showed enhanced
salt. Drought tolerance in sweet potato (Zhai et al., 2016) and over­
expression of Erianthus arundinaceus HSP70 (EaHSP70) showed
increased drought and salinity tolerance in sugarcane (Augustine et al.,
2015). In addition, MAPK2-MAPK4 was up-regulated under ROS ho­
meostasis (Pitzschke et al., 2009). These findings indicated that over­
expression of VuDREB2A indirectly affect the transcription of some of
the known drought- and heat-responsive genes which may add to
tolerance. Under drought stress, we also performed RNA-Seq of two
The authors sincerely acknowledge the financial support from the
Department of Biotechnology, Govt. of India, Centre of Excellence pro­
gram support (BT/402/NE/U-Excel/2013). We would like to express our
sincere gratitude to the Center for Application of Molecular Biology to
International Agriculture (CAMBIA), Australia, for pCAMBIA2301. We
also sincerely thank the Institute of Advanced Studies of Science and
Technology (IASST) for giving us access to use their instrumentation
facility. S.K sincerely acknowledges DST-SERB for the National post-
doctoral fellowship (PDF/2017/000761). S.K is also grateful for the
financial support from the DBT-RA Program in Biotechnology and Life
Sciences.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.plaphy.2022.09.028.
References
Agarwal, P.K., Jha, B., 2010. Transcription factors in plants and ABA dependent and
independent abiotic stress signaling. Biol. Plantarum 54 (2), 201–212.
Augustine, S.M., Narayan, J.A., Syamaladevi, D.P., Appunu, C., Chakravarthi, M.,
Ravichandran, V., Subramonian, N., 2015. Erianthus arundinaceus HSP70 (EaHSP70)
overexpression increases drought and salinity tolerance in sugarcane (Saccharum
spp. hybrid). Plant Sci. 232, 23–34.
Barnabas, B., Jäger, K., Fehér, A., 2008. The effect of drought and heat stress on
reproductive processes in cereals. Plant Cell Environ. 31 (1), 11–38.
Bastos, E.A., Nascimento, S.P., Silva, E.M., Freire Filho, F.R., Gomide, R.L., 2011.
Identification of cowpea genotypes for drought tolerance. Rev. Cienc. Agron. 42,
100–107.

12
S. Kumar et al.
Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for
water-stress studies. Plant Soil 39, 205–207.
Behnam, B., Kikuchi, A., Celebi-Toprak, F., Kasuga, M., Yamaguchi-Shinozaki, K.,
Watanabe, K.N., 2007. Arabidopsis rd29A:: DREB1A enhances freezing tolerance in
transgenic potato. Plant Cell Rep. 26 (8), 1275–1282.
Blum, A., 2005. Drought resistance, water-use efficiency, and yield potential— are they
compatible, dissonant, or mutually exclusive? Aust. J. Agric. Res. 56, 1159–1168.
Boo, Y.C., Jung, J., 1999. Water deficit—induced oxidative stress and antioxidative
defenses in rice plants. J. Plant Physiol. 155 (2), 255–261.
Boyer, J.S., 1982. Plant productivity and environment. Science 218 (4571), 443–448.
Chen, M., Wang, Q.Y., Cheng, X.G., Xu, Z.S., Li, L.C., Ye, X.G., Xia, L.Q., Ma, Y.Z., 2007.
GmDREB2, a soybean DRE-binding transcription factor, conferred drought and high-
salt tolerance in transgenic plants. Biochem. Biophys. Res. Commun. 353 (2),
299–305.
Chen, H., Liu, L., Wang, L., Wang, S., Cheng, X., 2016. VrDREB2A, a DREB-binding
transcription factor from Vigna radiata, increased drought and high-salt tolerance in
transgenic Arabidopsis thaliana. J. Plant Res. 129 (2), 263–273.
Chen, J.Q., Meng, X.P., Zhang, Y., Xia, M., Wang, X.P., 2008. Over-expression of OsDREB
genes lead to enhanced drought tolerance in rice. Biotechnol. Lett. 30 (12),
2191–2198.
Cong, L., Zheng, H.C., Zhang, Y.X., Chai, T.Y., 2008. Arabidopsis DREB1A confers high
salinity tolerance and regulates the expression of GA dioxygenases in Tobacco. Plant
Sci. 174 (2), 156–164.
Fleury, D., Jefferies, S., Kuchel, H., Langridge, P., 2010. Genetic and genomic tools to
improve drought tolerance in wheat. J. Exp. Bot. 61 (12), 3211–3222.
Fragkostefanakis, S., Röth, S., Schleiff, E., Scharf, K.-D., 2015. Prospects of engineering
thermotolerance in crops through modulation of heat stress transcription factor and
heat shock protein networks. Plant Cell Environ. 38 (9), 1881–1895.
Foyer, C.H., Noctor, G., 2005. Redox homeostasis and antioxidant signaling: a metabolic
interface between stress perception and physiological responses. Plant Cell 17 (7),
1866–1875.
Granier, C., Aguirrezabal, L., Chenu, K., Cookson, S.J., Dauzat, M., Hamard, P., Thioux, J.
J., Rolland, G., Bouchier-Combaud, S., Lebaudy, A., et al., 2006. PHENOPSIS, an
automated platform for reproducible phenotyping of plant responses to soil water
deficit in Arabidopsis thaliana permitted the identification of an accession with low
sensitivity to soil water deficit. New Phytol. 169, 623–635.
Gutierrez-Gonzalez, J.J., Guttikonda, S.K., Tran, L.S.P., Aldrich, D.L., Zhong, R., Yu, O.,
Nguyen, H.T., Sleper, D.A., 2010. Differential expression of isoflavone biosynthetic
genes in soybean during water deficits. Plant Cell Physiol. 51 (6), 936–948.
Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts: I. Kinetics and
stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198.
Ito, Y., Katsura, K., Maruyama, K., Taji, T., Kobayashi, M., Seki, M., Shinozaki, K.,
Yamaguchi-Shinozaki, K., 2006. Functional analysis of rice DREB1/CBF-type
transcription factors involved in cold-responsive gene expression in transgenic rice.
Plant Cell Physiol. 47 (1), 141–153.
Jeon, J.S., Jung, K.H., Kim, H.B., Suh, J.P., Khush, G.S., 2011. Genetic and molecular
insights into the enhancement of rice yield potential. J. Plant Biol. 54, 1–9.
Jia, X., Mao, K., Wang, P., Wang, Y., Jia, X., Huo, L., Sun, X., Che, R., Gong, X., Ma, F.,
2021. Overexpression of MdATG8i improves water use efficiency in transgenic apple
by modulating photosynthesis, osmotic balance, and autophagic activity under
moderate water deficit. Hortic. Res. 8, 81.
Kizis, D., Lumbreras, V., Pagès, M., 2001. Role of AP2/EREBP transcription factors in
gene regulation during abiotic stress. FEBS Lett. 498 (2–3), 187–189.
Kumar, S., Tanti, B., Patil, B.L., Mukherjee, S.K., Sahoo, L., 2017. RNAi-derived
transgenic resistance to Mungbean yellow mosaic India virus in cowpea. PLoS One
12 (10), e0186786.
Lata, C., Prasad, M., 2011. Role of DREBs in regulation of abiotic stress responses in
plants. J. Exp. Bot. 62 (14), 4731–4748.
Li, T., Huang, Y., Khadr, A., Wang, Y.H., Xu, Z.S., Xiong, A.S., 2020. DcDREB1A, a DREB-
binding transcription factor from Daucus carota, enhances drought tolerance in
transgenic Arabidopsis thaliana and modulates lignin levels by regulating lignin-
biosynthesis-related genes. Environ. Exp. Bot. 169, 103896.
Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K.,
Shinozaki, K., 1998. Two transcription factors, DREB1 and DREB2, with an EREBP/
AP2 DNA binding domain separate two cellular signal transduction pathways in
drought-and low-temperature-responsive gene expression, respectively, in
Arabidopsis. Plant Cell 10 (8), 1391–1406.
Lisei-de-Sá, M.E., Arraes, F., Brito, G.G., Beneventi, M.A., Lourenco-Tessutti, I., Basso, A.
M., et al., 2017. AtDREB2A-CA influences root architecture and increases drought
tolerance in transgenic cotton. Embrapa Recursos Genéticos e Biotecnologia-Artigo
em periódico indexado (ALICE). https://doi.org/10.4236/as.2017.810087.
Lv, S.L., Lian, L.J., Tao, P.L., Li, Z.X., Zhang, K.W., Zhang, J.R., 2009. Overexpression of
Thellungiella halophila H+-PPase (TsVP) in cotton enhances drought stress resistance
of plants. Planta 229, 899–910.
Magwanga, R.O., Lu, P., Kirungu, J.N., Lu, H., Wang, X., Cai, X., Zhou, Z., Zhang, Z.,
Salih, H., Wang, K., Liu, F., 2018. Characterization of the late embryogenesis
abundant (LEA) proteins family and their role in drought stress tolerance in upland
cotton. BMC Genet. 19 (1), 6.
Mizoi, J., Kanazawa, N., Kidokoro, S., Takahashi, F., Qin, F., Morimoto, K., Yamaguchi-
Shinozaki, K., 2019. Heat-induced inhibition of phosphorylation of the stress-
protective transcription factor DREB2A promotes thermotolerance of Arabidopsis
thaliana. J. Biol. Chem. 294 (3), 902–917.
13
Plant Physiology and Biochemistry 193 (2022) 1–13
Mizoi, J., Shinozaki, K., Yamaguchi-Shinozaki, K., 2012. AP2/ERF family transcription
factors in plant abiotic stress responses. Biochim. et Biophy. Acta (BBA)-Gene Regul.
Mech. 1819 (2), 86–96.
Morran, S., Eini, O., Pyvovarenko, T., Parent, B., Singh, R., Ismagul, A., Eliby, S.,
Shirley, N., Langridge, P., Lopato, S., 2011. Improvement of stress tolerance of wheat
and barley by modulation of expression of DREB/CBF factors. Plant Biotechnol. J. 9
(2), 230–249.
Nascimento, S.P., Bastos, E.A., Araújo, E.C.E., Freire Filho, F.R., Silva, E.M., 2011.
Tolerância ao déficit hídrico em genótipos de feijão-caupi. Rev. Bras. Eng. Agrícola
Ambient. 15, 853–860.
Oh, S.J., Kwon, C.W., Choi, D.W., Song, S.I., Kim, J.K., 2007. Expression of barley
HvCBF4 enhances tolerance to abiotic stress in transgenic rice. Plant Biotechnol. J. 5
(5), 646–656.
O’toole, J.C., Bland, W.L., 1987. Genotypic variation in crop plant root systems. In: Adv.
In Agro, vol. 41. Academic Press, pp. 91–145.
Panda, S.K., Singha, L.B., Khan, M.H., 2003. Does aluminium phytotoxicity induce
oxidative stress in greengram (Vigna radiata) Bulg. J. Plant Physiol. 29, 77–86.
Passardi, F., Cosio, C., Penel, C., Dunand, C., 2005. Peroxidases have more functions than
a Swiss army knife. Plant Cell Rep. 24 (5), 255–265.
Pitzschke, A., Djamei, A., Bitton, F., Hirt, H., 2009. A major role of the MEKK1–MKK1/
2–MPK4 pathway in ROS signaling. Mol. Plant 2, 120–137.
Powell, W., 2006. In: Ashraf, M., Harris, P.J.C. (Eds.), Abiotic Stresses: Plant Resistance
through Breeding and Molecular Approaches. The Haworth Press, Binghamton, NH,
USA, p. 370 (2005), pp. 725, US$89.95. ISBN 1-56022-965-9. Ex. Agric., 42370.
Qin, F., Kakimoto, M., Sakuma, Y., Maruyama, K., Osakabe, Y., Tran, L.S.P.,
Shinozaki, K., Yamaguchi-Shinozaki, K., 2007. Regulation and functional analysis of
ZmDREB2A in response to drought and heat stresses in Zea mays L. Plant J. 50 (1),
54–69.
Qu, Y., Sakoda, K., Fukayama, H., 2021. Overexpression of both Rubisco and Rubisco
activase rescues rice photosynthesis and biomass under heat stress. Plant Cell
Environ. 44 (7), 1–13.
Quan, L.J., Zhang, B., Shi, W.W., Li, H.Y., 2008. Hydrogen peroxide in plants: a versatile
molecule of the reactive oxygen species network. J. Integr. Plant Biol. 50 (1), 2–18.
Roorkiwal, M., Nayak, S.N., Thudi, M., Upadhyaya, H.D., Brunel, D., Mournet, P.,
This, D., Sharma, P.C., Varshney, R.K., 2014. Allele diversity for abiotic stress
responsive candidate genes in chickpea reference set using gene based SNP markers.
Front. Plant Sci. 5, 248.
Sadhukhan, A., Kobayashi, Y., Kobayashi, Y., Tokizawa, M., Yamamoto, Y.Y., Iuchi, S.,
Koyama, H., Panda, S.K., Sahoo, L., 2014. VuDREB2A, a novel DREB2-type
transcription factor in the drought-tolerant legume cowpea, mediates DRE-
dependent expression of stress-responsive genes and confers enhanced drought
resistance in transgenic Arabidopsis. Planta 240 (3), 645–664.
Sakuma, Y., Maruyama, K., Osakabe, Y., Qin, F., Seki, M., Shinozaki, K., Yamaguchi-
Shinozaki, K., 2006a. Functional analysis of an Arabidopsis transcription factor,
DREB2A, involved in drought-responsive gene expression. Plant Cell 18 (5),
1292–1309.
Sakuma, Y., Maruyama, K., Qin, F., Osakabe, Y., Shinozaki, K., Yamaguchi-Shinozaki, K.,
2006b. Dual function of an Arabidopsis transcription factor DREB2A in water-stress-
responsive and heat-stress-responsive gene expression. Proc. Natl. Acad. Sci. USA
103 (49), 18822–18827.
Saleh, A., Lumbreras, V., Lopez, C., Kizis, E.D.P.D., Pagès, M., 2006. Maize DBF1-
interactor protein 1 containing an R3H domain is a potential regulator of DBF1
activity in stress responses. Plant J. 46 (5), 747–757.
Schramm, F., Larkindale, J., Kiehlmann, E., Ganguli, A., Englich, G., Vierling, E., Von
Koskull-Döring, P., 2008. A cascade of transcription factor DREB2A and heat stress
transcription factor HsfA3 regulates the heat stress response of Arabidopsis. Plant J.
53 (2), 264–274.
Szabados, L., Savoure, A., 2010. Proline: a multifunctional amino acid. Trends Plant Sci.
15 (2), 89–97.
Wang, Q., Guan, Y., Wu, Y., Chen, H., Chen, F., Chu, C., 2008. Overexpression of a rice
OsDREB1F gene increases salt, drought, and low temperature tolerance in both
Arabidopsis and rice. Plant Mol. Biol. 67 (6), 589–602.
Wang, F., Liu, Y., Shi, Y., Han, D., Wu, Y., Ye, W., Yang, H., Li, G., Cui, F., Wan, S., Lai, J.,
Yang, C., 2020. SUMOylation stabilizes the transcription factor DREB2A to improve
plant thermotolerance. Plant Physiol. 183, 41–50.
Worch, S., Rajesh, K., Harshavardhan, V.T., Pietsch, C., Korzun, V., Kuntze, L., et al.,
2011. Haplotyping, linkage mapping and expression analysis of barley genes
regulated by terminal drought stress influencing seed quality. BMC Plant Biol. 11 (1),
1–14.
Yamaguchi-Shinozaki, K., Shinozaki, K., 1994. A novel cis-acting element in an
Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-
salt stress. Plant Cell 6 (2), 251–264.
Zhai, H., Wang, F., Si, Z., Huo, J., Xing, L., An, Y., He, S., Liu, Q., 2016. A myo-inositol-1-
phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem
nematode resistance in transgenic sweet potato. Plant Biotechnol. J. 14 (2),
592–602.
Zhao, J., Ren, W., Zhi, D., Wang, L., Xia, G., 2007. Arabidopsis DREB1A/CBF3 bestowed
transgenic tall fescue increased tolerance to drought stress. Plant Cell Rep. 26 (9),
1521–1528.
Zhou, Y., Chen, M., Guo, J., Wang, Y., Min, D., Jiang, Q., et al., 2019. Overexpression of
soybean DREB1 enhances drought stress tolerance of transgenic wheat in the field.
J. Exp. Bot. 71, 1842–1857.