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Grapevine RD22a constitutive expression in tobaccoenhances stomatal adjustment and confers droughttolerance
Rahma Jardak-Jamoussi . Donia Abdelwahed . Nejia Zoghlami . Asma Ben Salem .
Olfa Zarrouk . Ahmed Mliki . Manuela Chaves . Abdelwahed Ghorbel . Carla Pinheiro
Received: 23 August 2016 / Accepted: 22 September 2016 / Published online: 4 October 2016
� Brazilian Society of Plant Physiology 2016
Abstract Drought is one of the major constraints
limiting crop production worldwide including grape-
vine. Investigations of drought tolerance genotypes by
genetic engineering are an important goal in Vitis
breeding program. Three dehydration-responsive
RD22 genes (VviRD22) were identified in Vitis
vinifera L. Here, we aim to evaluate the constitutive
expression of VviRD22a effect on tobacco perfor-
mance under low water availability conditions,
namely under drought and under osmotic stress.
In vitro, and under osmotic stress, transgenic seeds
of tobacco showed an enhanced tolerance at the
germination and seedling stages compared to the wild-
type (WT). When drought was applied ex vitro by
stopping irrigation during 9 days, transgenic lines
exhibited an earlier decrease of stomatal conductance
that was, interestingly, followed by an internal adjust-
ment leading to a moderate decline of the photosyn-
thetic rate. Additionally, differences between WT and
transgenics under both control and stressed conditions
were revealed at ultrastructural level through shape
alteration within the transgenics. Additionally, the
performances of the VviRD22a lines under drought
were notably maintained in terms of biomass produc-
tion (vegetative dry material) and water status (Relative
water content and water retention ability). A significant
distinctiveness between VviRD22a-expressing lines
and WT under stress conditions but not under control
conditions (principal component analyses) was found.
Protection effect of VviRD22a constitutive expression
towards drought involved root biomass, water status
and stomatal adjustment traits. Overall, our data suggest
that VviRD22a transgenic expression plays a positive
role in drought tolerance improvement supporting it as
an important candidate gene for molecular breeding of
drought tolerant grapevines.
Keywords Constitutive gene expression � Droughttolerance improvement � Germination � Stomatal
adjustment
1 Introduction
Faced with scarcity of water resources, drought is the
most critical threat to important field crops. This
Electronic supplementary material The online version ofthis article (doi:10.1007/s40626-016-0077-3) contains supple-mentary material, which is available to authorized users.
R. Jardak-Jamoussi (&) � D. Abdelwahed �N. Zoghlami � A. Ben Salem � A. Mliki � A. GhorbelLaboratory of Plant Molecular Physiology, Biotechnology
Center of Borj Cedia, BP901, 2050 Hammam-Lif, Tunisia
e-mail: [email protected]
O. Zarrouk � M. Chaves � C. PinheiroPlant Molecular Ecophysiology Lab, ITQB, NOVA,
Av. da Republica, 2780-157 Oeiras, Portugal
C. Pinheiro
Faculdade de Ciencias e Tecnologia, Universidade NOVA
de Lisboa, 2829-516 Caparica, Portugal
123
Theor. Exp. Plant Physiol. (2016) 28:395–413
DOI 10.1007/s40626-016-0077-3
Author's personal copy
constraint impairs normal plant growth, disturbs water
relations, and reduces water use efficiency in plants
(Farooq et al. 2009). Grapevine (Vitis vinifera L.) is one
of the most widely cultivated and economically
important fruit crop in the world. With the global
warming, its growth and yield are predicted to be
dramatically affected by drought (Serra et al. 2013).
Among the measures believed to prevent drought-
caused damages, the cultivation of stress tolerant plants
has been considered to be the most promising
(Nakashima and Yamaguchi-Shinozaki 2005; Valliyo-
dan and Nguyen 2006). In this context, biotechnology
offers promise as a means of improving food security
and reducing pressures on the environment, provided
the perceived environmental threats from biotechnol-
ogy itself are addressed. Genetically modified crop
varieties resistant to drought could help to sustain
farming in marginal areas and to restore degraded lands
to production. In this context, development of crop
plants tolerant to drought stress was found the potential
approach that would help to guarantee stable produc-
tivity. To attempt this goal, valuable work has been
done on drought tolerance in plants and efforts were
made to understand the physiological mechanisms and
genetic control of the contributing traits at different
plant developmental stages (Hasegawa et al. 2000; Hu
and Xiong 2014; Sun et al. 2016; Hu et al. 2010).
Therefore, specific aspects were elucidated in plants
responses to cope with this constraint, including
stomata adjustment, osmo-regulation, selective uptake,
ion compartmentation, etc. (Agarwal et al. 2006; Penna
2003; Reddy et al. 2011; Blumwald 2000). With the
aim to produce drought tolerant and more productive
lines, plant breeding is being used since long. However,
the exploitation of these programs is limited due to
multigenic nature of drought tolerance and presence of
low genetic variation inmajor crops (Turan et al. 2012).
The biotechnological tools and genetic engineering
constitute an efficient strategy for achieving enhanced
plant drought tolerance (Tardieu 2010; Cushman and
Bohnert 2000; Hu and Xiong 2014; Deng and Dong
2013; Zhang et al. 2008). Blum (2014) reported that
insufficient phenotyping of experimental transgenic
plants for drought resistance often does not allow true
conclusions about the real function of the discovered
genes towards drought resistance. So an outline of a
minimal set of tests would help to resolve the valid
utility of revealed genes, thus bringing the research
results down to earth.
Within Vitis vinifera, the development of drought
tolerant genotypes is an important breeding goal since
genetic transformation was used as a key technology
to enhance resistant cultivars to abiotic stress (Jardak-
Jamoussi et al. 2009; Gambino et al. 2010). In this
context, the physiological, biochemical, genetic and
metabolic mechanisms to tolerate water constraints
were explored (Hochberg et al. 2013; Lovisolo et al.
2010; Cramer et al. 2007; Chaves and Oliveira 2004).
Microarray analyses of water stressed ‘Cabernet
Sauvignon’ grapevines demonstrated that more than
2000 genes were differentially expressed, and expres-
sion was influenced by both drought and abscisic acid
(ABA) (Cramer et al. 2007). With this tremendous
amount of information accumulated through genome
mining and expression analysis, more critical genes/
promoters are revealed and become available for use
(Gray et al. 2014; Cramer et al. 2013; Cramer 2010).
Nevertheless, the contribution for drought tolerance
enhancement is only available for a few of them
(Perrone et al. 2012; Cramer 2010; Gray et al. 2014;
Jardak-Jamoussi et al. 2016).
A grapevine RD22 genewas identified (Hanana et al.
2008), which is constitutively expressed in all tissues
and its expression was induced by drought and salt
stress. TheRD22genewas reported to be a stress-related
gene which induction is mediated by ABA and requires
activation by transcription factors such as MYC and
MYB (Abe et al. 1997; 2003). Cramer et al. (2007);
Deluc et al. (2007); Espinoza et al. (2007) reported the
possible existence of other RD22 genes in grape by
microarrays experiments in different grapevine organs.
In this context,Matus et al. (2014) identified and studied
three Vitis RD22 genes (VviRD22a, VviRD22b and
VviRD22c) from Cabernet Sauvignon. The grapevine
RD22 gene previously isolated by Hanana et al. (2008)
was named byMatus et al. (2014) VviBURP05 and was
referenced as VviRD22a. The three identified genes
shared 50–70 % similarity in their complete protein
sequences and over 90 % similarity in their BURP
domain (a domain found at the C terminus of several
other plant proteins such as USP embryonic abundant
and polygalacturonase proteins (BURP: BNM2, USP,
RD22, PG1b).TheVviRD22a andbgeneswere induced
by salt treatments of nodal segments but VviRD22cwas
inhibited. The constitutive VviRD22a expression (Ac-
cessionNo.AY634282) in the tobacco plantswas found
to improve salt tolerance fromgermination to adult plant
stage (Jardak Jamoussi et al. 2014). We established as
396 Theor. Exp. Plant Physiol. (2016) 28:395–413
123
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working hypothesis that the constitutive expression of
RD22 alters the pattern of stomata opening and there-
fore plant performance and biomass partitioning. In
order to test this hypothesis, we compared the physio-
logical responses of two T2 VviRD22a-expressing lines
and wild-type (WT) tobacco plants subjected to water
deficit using in vitro and ex vitro assays. This evaluation
was based on the germination aptitude, stomata pattern
and functioning, photosynthetic parameters, biomass
production and water status.
2 Materials and methods
2.1 Plant material
The T2 Nicotiana benthamiana Domin seeds of the
L15 and L20 transgenic lines and WT (Jardak
Jamoussi et al. 2014) were used for the in vitro
germination assays on Murashige and Skoog (MS)
medium (1962), MS plates and for the greenhouse
assays (water stress assay). Seeds were incubated at
4 �C for 3 days to promote synchronous germination
and growth at 25 �C.
2.2 In vitro germination assays
Fifty seeds from WT and transgenic lines were
cultivated in Petri dishes. Seeds were scored as
germinated when the radicle tips had fully expanded
the seed coat. The percentage of germinated seeds was
scored as the germination success. The sensitivity of T2
seed germination to Mannitol was assayed on MS
medium agar plates containing 0, 200 and 300 mM
Mannitol. The sensitivity to PEGwas tested forWTand
transgenic seeds placed on filter paper with MS liquid
medium including 0, 5, 10 and 15 %PEG.Germination
sensitivities to Mannitol and PEG were scored every
three days for threeweeks as ratios of stressed to control
germinated seeds. Three replicates were done for each
treatment. Germination was carried out under con-
trolled room conditions (24-25 �C temperature, 16 h
photoperiod and 70 lmol m-2 S-1 light intensity).
2.3 Greenhouse assay
Greenhouse experiments were established in order to
assess potential drought tolerance of VviRD22a-
expressing tobacco plants. After in vitro germination
on MS medium containing 200 mg L-1 kanamycin,
seedlings were acclimatized in the greenhouse under a
light intensity of 25 Wm-2, an average temperature of
258/18 �C and a relative humidity of 60–70 % for two
week. The plants were then individually transplanted
in pots filled with sandy soil and regularly irrigated to
the field capacity (FC) with a diluted Long Ashton
nutrient solution (Hewitt 1966) for one month. Sub-
sequently, for both WT and transgenic L15 and L20
lines, drought was applied by withholding irrigation
during nine days (control plants, WT and transgenic,
continued to be irrigated to the field capacity at 3 days
intervals). A lethal effect of dehydration was observed
on most of WT after nine days drought, while
transgenics were able to recover. Triplicates were
performed for all lines, data points and evaluated
parameters.
2.3.1 Growth parameters
Shoot and root fresh weight from WT and transgenic
tobacco plant were determined under control and
stress conditions at the end of the drought assay (9th
day). Subsequently, the corresponding dry biomasses
were measured after oven drying for 48 h at 70 �C.
2.3.2 Stomatal conductance and photosynthetic
assimilation
Stomatal conductance gs (mol m-2 s-1) and photo-
synthetic assimilation Amax (lmol m-2 s-1) rates
were measured on a young and fully developed leaf
from WT and transgenic (L15 and L20) plants, using
an automated photosynthetic measuring apparatus
LCpro operating at 25 �C temperature, 60 % air
humidity, 365 ppm [CO2] and 1000 lmol m-2 s-1
PPFD. The gs and Amax values were registered during
the morning (8 to 10 am) on 3 replicates per treatment,
at the first, 3rd, 6th and 9th day of water stress. Plants
were grown under light energy of 25Wm-2 and 16/8 h
light/dark photoperiod.
2.3.3 Stomata shape
Small leaf samples were collected from WT and
transgenic lines under control and water stress condi-
tions at the end of assay (9th day) and directly inserted
inside an Environmental Scanning Electron Micro-
scopy SEM (Quanta 200 FEI, FEI Company,
Theor. Exp. Plant Physiol. (2016) 28:395–413 397
123
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Hillsboro, OR) for fresh biological material observa-
tion. Backscattered Electron images (BSE) were taken
at an accelerating voltage of 15, 3 kV and a vapour
pressure of 0.68 Torr.
2.3.4 Leaf water parameters
Leaf relative water content (RWC) was estimated in
sets of 10 leaf discs of 7 mm diameter and similar
physiological stage from all plants were used to
estimate the relative water contents, as follows:
RWC (%) = [(FW-DW)/(TW-DW)] 9 100;
where TW is the turgid weight.
Water retention ability (WRA) was estimated in
leaves that were weighed (fresh weight), then desic-
cated for 24 h under controlled conditions (65 %
relative humidity and 25 �C), prior to be weighed
again (desiccated weight). The leaves were finally
oven-dried during 48 h at 70 �C to a constant dry
weight. Water retention ability was calculated accord-
ing to Jia et al. (2008) as follows:
WRA (%) = [(desiccated weight-dry weight)/
(fresh weight-dry weight)] 9 100.
2.3.5 Osmotic potential
Disks of fully expanded adult leaf (5 mm) from
control and water stressed of WT and transgenic
tobacco plants were excised in the morning at the end
of the assay. Following the method of (Martınez-
Ballesta et al. 2004), the resulting sap was analyzed for
osmolarity determination. Osmolarity was assessed
using an osmometer (OSMOMAT 030) and converted
from mOsmol kg-1 to MPa to determine the osmotic
potential (Ws) according to the Van’t Hoff equation:
Ws = -m 9 R 9 T, where m is the osmolality, R the
universal gas constant, and T is the temperature (K).
2.4 Statistical analysis
Data are means of three replicates from three different
plants from control and stressed sets. The results were
analyzed by comparing (F) values obtained from one-
way ANOVA (Fast statistics v 2.0.4). Whenever
significant interaction between genotypes and treat-
ments were found (GXT), the least significant differ-
ences (LSD; p\ 0.05) were calculated using
STATISTICA software.
Principal component analysis (PCA) followed
between groups analysis (BGA) was performed using
the R platform (version 2.13.1, R Development Core
Team, 2011) and the ade4TkGUI package was used
(Thioulouse and Dray 2009). The Pearson’s product
moment correlation coefficient was calculated using
the cor.test in order to disclose significant relation-
ships between principal components and the variables
analysed (p\ 0.05).
3 Results
3.1 Effect of water stress on VviRD22a and wild-
type tobacco plants under in vitro conditions
Seeds of WT and transgenic L15 and L20 lines were
tested for germination on MS medium with Mannitol
or PEG (6000) at different concentrations.
Germination ratios (stressed to control: ST/C) in the
presence of 200 and 300 mM Mannitol showed
significant difference for each genotype across all
dates (Table 1, Supplemental Table 1), the lines L15
and L20 exhibiting a better performance. It was clear
that in the presence of 200 mM Mannitol, the germi-
nation at the 3rd day both in WT and L20 lines was
absent and was at low rate for L15. However,
significant differences started to be observed among
lines beginning of the 6th day. In fact, stressed to
control ratios of success germination of L15 and L20
were of 0.62 ± 0.06 and 0.88 ± 0.02 respectively.
However, WT seeds did not show germination at this
date. These high ratios would be attributed to
VviRD22a expression. Additionally, at the 9th day,
L15 and L20 ratios were increased significantly
compared to WT which exhibited very low ratio
(0.1 ± 0.01) that indicate that germination was sig-
nificantly inhibited (Table 1).
Along the germination period (12th, 15th, 18th and
21th days), transgenic seeds ratios at the 12th day
attained 1 ± 0.0 and 1 ± 0.009 for L15 and L20
respectively implying that germination was not
affected. In case of WT, germination was delayed
and corresponding ratio was significantly lower com-
pared to transgenics (0.86 ± 0.008) (Table 1). Under
300 mM Mannitol, WT seeds germination ratios was
significantly lower and did not exceed 0.14 ± 0.009
while germination ratios attained by transgenic lines
was not so severely decreased (0.29 ± 0.006 and
398 Theor. Exp. Plant Physiol. (2016) 28:395–413
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0.44 ± 0.006 after 21 days, within L15 and L20 lines
respectively). Moreover, germination on 300 mM
Mannitol supplemented MS medium reveal a distinc-
tion between L15 and L20 transgenic lines (Table 1).
In fact, germination of L20 started at the day 6 and
displayed ST/C ratio of 0.07 ± 0.003, while L15
started germination at the 12th day treatment. Addi-
tionally, at the day 21, L20 exhibited a ST/C ratio
significantly different from L15. Both, L15 and L20
seedlings were able to growth on MS containing
300 mM Mannitol (Supplemental figure S1). Thus,
transgenic seeds exhibited significantly higher green
cotyledon percentages at 300 mM Mannitol (14 ± 1
and 17 ± 3 % in L15 and L20, respectively). How-
ever, within WT, green cotyledons emergence was
almost absent (1.7 ± 0.5 %) (Fig. 1a).
Water stress induced by PEG was also found to
show significant differences. Statistical analysis by
One-way ANOVA showed that differences for each
genotype were significant across all dates (from the
3rd to the 21th day treatment) and all treatments (5, 10
and 15 % PEG). When comparing all genotypes
across all dates, 5 % PEG was the most discriminative
concentration (Table 2; Supplemental Table 2)
between all genotypes, since corresponding F-ratio
obtained for this treatment (5 % PEG) was the highest
(17.1303 vs. 10.4539 and 10.2935 for 10 and 15 %
PEG respectively).
Germination rates of WT and transgenic seeds
cultivated on 5 % PEG supplemented MS media
(Table 2) revealed significantly, from the 3rd day on,
higher germination ratios in L15 and L20 transgenic
lines than in WT. When water stress was induced by
10 % PEG, transgenic lines start germination from the
3rd day with significant difference in ratios up to the 9th
day. However, WT showed germinated seeds from the
6th day. At this concentration, significant difference in
ratios betweenWT and transgenic was clear till 21 days
cultivation. When PEG concentration was increased to
15 %, transgenic lines exhibited significantly higher
ratios from the 6th day of cultivation up to 21st day than
WT which start germination only at the 9th day. The
15 % PEG supplementedMSmedium allow distinction
between the lines L15 and L20 up to 18th day.
Germination experiments allowed to notice higher
percentages of green cotyledons in transgenic lines
Table 1 Comparison of in vitro germination ratios (stress/control: ST/C) of WT and VviRD22a- expressing tobacco plants (lines
L15 and L20) during the drought till the 21th day on MS medium including Mannitol at 200 and 300 mM
200 mM mannitol ratio ST/C 300 mM mannitol ratio ST/C
Day WT L15 L20 WT L15 L20
3 0 ± 0.00 0.004 ± 0.003 0 0 ± 0.00 0 ± 0.00 0 ± 0.00
6 0 ± 0.00 0.62 ± 0.06f 0.88 ± 0.02cde 0 ± 0.00 0 ± 0.00 0.007 ± 0.003h
9 0.1 ± 0.01h 0.82 ± 0.05e 0.89 ± 0.04bcde 0 ± 0.00 0 ± 0.00 0.007 ± 0.003h
12 0.41 ± 0.003g 1 ± 0.007abcd 1 ± 0.00a 0.02 ± 0.003h 0.07 ± 0.003g 0.15 ± 0.00e
15 0.8 ± 0.09e 0.97 ± 0.007abcd 1 ± 0.00ab 0.12 ± 0.01f 0.16 ± 0.024e 0.23 ± 0.007d
18 0.8 ± 0.01e 0.97 ± 0.003abcd 1 ± 0.00ab 0.13 ± 0.01ef 0.25 ± 0.006d 0.37 ± 0.006b
21 0.86 ± 0.01de 1 ± 0.01abc 1 ± 0.00ab 0.14 ± 0.008ef 0.29 ± 0.02c 0.44 ± 0.006a
One-way Anova
All genotypes across all dates
F-ratio 7.4028 4.1469
F-critical 3.1504 3.1504
Genotype 9 drought
F-ratio[F-critical Yes Yes Yes Yes Yes Yes
All genotypes across all treatments
F-ratio 4.7789
F-critical 3.0699
Data are mean ± SE of 3 replicates. One-way ANOVA was preformed to detect significant interactions between genotypes and
treatments. When found significant, the least significant differences (LSD) were calculated
The letters indicate significant differences according to LSD test (p B 0.05)
Theor. Exp. Plant Physiol. (2016) 28:395–413 399
123
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than WT compared to controls on MS including 0, 5,
10 and 15 % PEG (Supplemental figure S2). In fact,
under 10 % PEG (Fig. 1b, Supplemental figure S2),
we observed that L15 and L20 seedlings continued
their growth and exhibited an optimal development
(95 ± 1 and 99 % respectively for L15 and L20).
However, in WT less green cotyledon rates were
exhibited (40 ± 1 %). Under 15 % PEG, the trans-
genic seeds kept their aptitude for green cotyledon
development, however for WT seeds green cotyledon
rates decreased and growth stopped (Supplemental
figure S2). The transgenic seeds ability for seedling
development would be related to VviRD22a constitu-
tive expression.
When comparing PEG and Mannitol treatments by
analyzing all genotypes across all treatments, PEG’s
F-ratio (10.4012) was higher than that of Mannitol
(4.7789). This reveals that PEG induces relevant
distinction than Mannitol for germination capacity
(Tables 1, 2; Supplemental Tables 1, 2).
3.2 Effect of water stress on VviRD22a tobacco
under controlled greenhouse conditions
Since VviRD22a expression in tobacco enhanced
germination and seedlings growth under in vitro water
stress conditions, it was crucial to investigate its
eventual effect on the physiological responses under
greenhouse conditions.
3.2.1 Photosynthetic parameters of WT
and transgenic tobacco lines under drought
Photosynthetic evaluation of WT and transgenic
plants was based on stomatal conductance (gs) and
net photosynthetic assimilation (Amax) measurement
of control and water-stressed plants (Tables 3, 4).
After 4 weeks acclimation under controlled green-
house conditions, no phenotypic differences were
observed between VviRD22a expressing and WT
plants under control conditions. This indicates that
the ectopic expression of VviRD22a does not affect the
overall plant morphology and biomass production
(Fig. 2a).
Under control conditions and day 1, gs and Amax
(Tables 3, 4) rates were not significantly different
between WT and transgenic lines. When subjected to
drought, gs (Table 3, Supplemental Table 3) of trans-
genic lines were significantly lower than those of WT,
since the 3rd day of stress. This time point showed, by
one-way ANOVA analysis, the highest F-ratio (10.
8773) than the others when comparing all genotypes.
In fact, 31.2 ± 11.62 and 24.32 ± 5.25 % gs
decreases were respectively registered in L15 and
L20 compared to controls on day 3, whereas inWT the
decrease in gs was about 7.4 ± 1.8 % compared to
control (Table 3: genotype vs. stress). However, on
the 6th day of drought, stress effect was not significant
in L15 and L20 in contrast to WT. In fact, the decrease
in gs within L15 and L20 lines was about 29 ± 6,
34 ± 5 and 48 ± 19 % respectively in L15, L20 and
WT. Significant difference was observed only for WT
(Table 3, Supplemental Table 3: Genotype vs. stress)
and significance between all genotypes remains high
at this stress time point (6.7425). Evenly, exhibition of
same behaviour at the 9th day of water stress was also
observed with the highest significance within WT
a a a
dc b
0
20
40
60
80
100
120
WT L15 L20
Gre
en se
edlin
gs (%
)MS
300 mM Mannitola
a a a
c
b a
0
20
40
60
80
100
120
WT L15 L20
Gre
en se
edlin
gs (%
)
MS
10% PEGb
Fig. 1 In vitro green seedlings on a Mannitol-free MS and
300 mM Mannitol supplemented MS medium and on
b PEG6000 free MS and on PEG6000 presence at 10 %. Data
are mean ± SE of 3 replicates. The letters indicate significant
differences according to LSD test (p B 0.05)
400 Theor. Exp. Plant Physiol. (2016) 28:395–413
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Table
2Comparisonofin
vitro
germinationratios(stress/control:ST/C)ofWTandVviRD22a-expressingtobacco
plants(lines
L15andL20)duringthedroughttillthe21th
day
onMSmedium
includingPEG
at5,10and15%
Day
ofculture
5%
PEG_Ratio
ST/C
10%
PEG_Ratio
ST/C
15%
PEG_Ratio
ST/C
WT
L15
L20
WT
L15
L20
WT
L15
L20
30.505±
0.05f
0.711±
0.03e
0.72±
0.04e
0±
0.00
0.12±
0.01j
0.045±
0.01k
0±
0.00
0±
0.00
0±
0.00
60.764±
0.01e
1±
0.00a
0.92±
0.02bc
0.57±
0.006gh
0.94±
0.01de
0.74±
0.01f
0±
0.00
0.02±
0.006k
0.08±
0.01ij
90.745±
0.01e
1±
0.00a
0.94±
0.02bc
0.55±
0.003hi
0.95±
0.01bcd
0.92±
0.02e
0.06±
0.01j
0.47±
0.009c
0.14±
0.009h
12
0.753±
0.03e
1±
0.00a
0.98±
0.005ab
0.53±
0.01i
0.97±
0.01abcd
0.95±
0±
008cde
0.1
±0.005hi
0.51±
0.01b
0.18±
0.01g
15
0.85±
0.03d
1±
0.00a
1±
0.00a
0.55±
0.02hi
0.98±
0.01abc
0.97±
0.01abcd
0.19±
0.007g
0.52±
0.006ab
0.3
±0.03e
18
0.887±
0.01cd
1±
0.00a
1±
0.00a
0.56±
0.01hi
0.98±
0.01abc
0.99±
0.003ab
0.24±
0.007f
0.53±
0.00ab
0.41±
0.02d
21
0.897±
0.01cd
1±
0.00a
1±
0.00a
0.6
±0.02g
1±
0.003a
1±
0.003a
0.24±
0.01f
0.54±
0.02a
0.51±
0.02ab
One-way
Anona
Allgenotypes
across
alldates
F-ratio
17.1303
10.4539
10.2935
F-critical
3.1504
3.1504
3.1504
Genotype9
drought
F- ratio[
F-
critical
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Allgenotypes
across
alltreatm
ents
F-ratio
10.4012
F-critical
3.0445
Dataaremean±
SEof3replicates.One-way
ANOVA
was
preform
edto
detectsignificantinteractionsbetweengenotypes
andtreatm
ents.When
foundsignificant,theleast
significantdifferences(LSD)werecalculated
Thelettersindicatesignificantdifferencesaccordingto
LSD
test(p
B0.05)
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(Table 3, genotype vs. stress, F-ratio: 26.2628). In fact
gs decrease was around 37 ± 4, 40 ± 3 and
61 ± 14 %, respectively for L15, L20 and WT
respectively) (Table 2). At this stress time point (9th
day) distinction between both transgenics was
observed (Table 3, genotype versus stress).
Nevertheless gs decreased more in both transgenic
lines than WT at the 3rd day of stress, later up to the
end of the assay (6th and 9th days’ stress) the highest
levels were measured in the transgenics. The gs
measurement would indicate an early stomatal control
within the transgenic lines and a water saving strategy.
Regarding photosynthetic rates (Amax), significant
differences were observed in transgenic lines and WT
under drought compared to control at 3rd, 6th and 9th
stress days (Table 4, Supplemental Table 4-1). A
decrease of Amax (about 31 ± 11 and 35 ± 20 %),
was recorded in the transgenic lines at the 3rd day.
Whereas, a more significant Amax decrease
(51 ± 17 %) was observed in WT. At the 6th days
of drought, Amax decreased by about 38 ± 6 and
44 ± 6.8 % and at the 9th day, the decrease was
64 ± 6 and 66 ± 4 % in the transgenic lines
respectively; whereas; in WT, a greater decline was
observed (75 ± 6 %). Through one-way ANOVA
analyses, the higher significance difference across all
genotypes was at the 9th day of stress. The transgenic
lines would exhibit a physiological adjustment due to
precocity in stomatal control. Also, comparing gs and
Amax across all genotypes and across all dates, Amax
was more relevant for distinction (Supplemental
Table 4-2).
3.2.2 Water status and osmotic adjustment
To analyse the water status of transgenic plants under
drought, the relative water content (RWC), water
retention ability (WRA) and osmotic potential (W)
were measured at the end of the assay (after 9 days of
water stress; Table 5, Supplemental Table 5).
Under control conditions, leaf relative water con-
tents (Table 5) were significantly higher in WT
(93 ± 0.4 %) than in transgenic L15 (82 ± 6 %)
and L20 (73 ± 3 %) lines.When subjected to drought,
much more significant decline in RWC was registered
in WT (46 ± 6 %) than in transgenic lines
Table 3 Comparison of ex vitro stomatal conductance (gs) of WT and VviRD22a- expressing tobacco plants (lines L15 and L20)
during the drought (day 1, day 3, day 6 and day 9)
gs F-ratio[F-critical
Day 1 Day 3 Day 6 Day 9
WT-C 0.54 ± 0.02 0.52 ± 0.01a 0.55 ± 0.03a 0.49 ± 0.04 Yes F-ratio 6.1442
WT-ST 0.6 ± 0.02 0.51 ± 0.005ab 0.28 ± 0.04de 0.18 ± 0.03 F-critical 4.3009
L15-C 0.6 ± 0.05 0.62 ± 0.05a 0.53 ± 0.02a 0.52 ± 0.09 Yes F-ratio 11.2771
L15-ST 0.56 ± 0.05 0.43 ± 0.04bc 0.38 ± 0.07 cd 0.31 ± 0.03 F-critical 4.3009
L20-C 0.63 ± 0.07 0.58 ± 0.04a 0.52 ± 0.05a 0.53 ± 0.06 Yes F-ratio 9.6824
L20-ST 0.6 ± 0.01 0.44 ± 0.02bc 0.34 ± 0.02 cd 0.3 ± 0.05 F-critical 4.3009
One-way Anona
All genotypes
F-ratio 0.5735 10.8773 6.7425 0.3075 5.4375
F-critical 3.1059 3.1059 3.1059 3.1059 2.3538
WT 9 Stress
F-ratio[F-critical No No Yes Yes
L15 9 Stress
F-ratio[F-critical No Yes No No
L20 9 Stress
F-ratio[F-critical No Yes No Yes
Data are mean ± SE of 3 replicates. One-way ANOVA was preformed to detect significant interactions between genotypes and
treatments. When found significant, the least significant differences (LSD) were calculated
The letters indicate significant differences according to LSD test (p B 0.05)
402 Theor. Exp. Plant Physiol. (2016) 28:395–413
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(14.6 ± 5.7 and 15.5 ± 2.1 % in L15 and L20,
respectively compared to control).
For water retention abilities (WRA) no marked
declines were registered in transgenic lines compared
to corresponding controls (Table 5, Supplemental
Table 5). However, compared to WT significant
differences were registered. Indeed, a decrease of
37 ± 9 % was recorded in WT, whereas it did not
exceed 6 ± 2 and 8 ± 1 % in L15 and L20 lines
respectively. These results reflect ability of VvRD22a-
expressing plants to cope water loss as a stress
avoidance strategy, when subjected to drought. At
the phenotypic level, more wilted leaves were
observed among WT plants, however, leaves in
transgenic lines were similar to control plants (Fig. 2).
Osmotic potential analysis confirms this strategy as
after 9 days of drought, L15 and L20 but not WT
exhibited significantly more negative osmotic poten-
tial. Nevertheless, across all genotypes, RWC and to a
lesser extent WRA, were found to be significantly
affected (one-way ANOVA, Table 5; Supplemental
Table 5). L15 and L20 distinction (genotype x stress),
was revealed through WRA and osmotic potential
since significance of decline of WRA was registered
only in L20, while diminution of osmotic potential
was significant only in L15.
3.2.3 Scanning electron microscopy
Under control conditions, a difference in stomata
shape was observed by SEM between WT (Fig. 3,
right panel) and transgenic lines (Fig. 3 left panel).
L15 line clearly exhibited an elliptical stomata shape
compared to WT (ovoid shape). This stomatal ultra-
structural alteration in the transgenic lines can be
related with the better water status and higher
photosynthetic capacity (Saibo et al. 2009).
3.2.4 Shoot and root biomasses
To quantify the phenotypic differences between WT
and VvRD22a-expressing lines grown under drought
conditions, fresh and dry biomasses of tobacco shoots
and roots were measured in order to seek for contri-
bution of VviRD22a expression in biomass mainte-
nance under drought.
Table 4 Comparison of photosynthetic activity (Amax) of WT and VviRD22a- expressing tobacco plants (lines L15 and L20) during
the drought (day 1, day 3, day 6 and day 9)
Amax F-ratio[F-critical
Day 1 Day 3 Day 6 Day 9
WT-C 36.19 ± 2.75 31.53 ± 2.76a 30.15 ± 3.89a 30.52 ± 3.18a Yes F-ratio 18.2352
WT-ST 33.98 ± 2.89 14.82 ± 1.27de 13.35 ± 0.31e 7.51 ± 0.12f F-critical 4.3009
L15-C 37.71 ± 2.44 35.22 ± 0.27a 32.57 ± 0.98a 33.52 ± 1.37a Yes F-ratio 18.6546
L15-ST 35.43 ± 0.96 24.04 ± 1.78b 20.18 ± 1.5bc 12.12 ± 0.66e F-critical 4.3009
L20-C 36.95 ± 3.82 36.56 ± 3.74a 34.04 ± 2.61a 37.43 ± 1.82a Yes F-ratio 24.1372
L20-ST 23.15 ± 2.33 18.65 ± 2.39b 18.65 ± 0.35cd 12.04 ± 0.29e F-critical 4.3009
One-way Anova
All genotypes
F-ratio 0.3075 12.9181 17.1861 64.3149 13.1212
F-critical 3.1059 3.1059 3.1059 3.1059 2.3538
WT 9 Strss
F-ratio[F-critical No Yes Yes Yes
L15 x Stress
F-ratio[F-critical No Yes Yes Yes
L20 x Stress
F-ratio[F-critical No Yes Yes Yes
Data are mean ± SE of 3 replicates. One-way ANOVA was preformed to detect significant interactions between genotypes and
treatments. When found significant, the least significant differences (LSD) were calculated
The letters indicate significant differences according to LSD test (p B 0.05)
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123
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Under control conditions, WT and both transgenic
lines did not display any phenotypic variability and no
significant differences in shoot (aerial part) and root
fresh (FW) and dry weights (DW) (Table 6) were
observed. When subjected to drought stress, both in
WT and transgenic plants shoot and root FW decrease
significantly compared to the corresponding controls,
but twice as more in WT (Table 6, Supplemental
Table 6). However, in VviRD22a-expressing lines,
shoot and root DW at the 9th day stress did not show
significant decline which was in contrast to WT
(Table 6, Supplemental Table 6). In fact, the declines
in shoot DW inWT was 3 and 1.8 fold higher than that
in L15 and L20 respectively. For root DW, the
transgenic lines were able to keep higher weights
compared to WT (Table 6). Comparison of root and
shoot DW/FW ratios within and across all genotypes
did not reveal any significant difference. In what
concern leaf number, we observe under control
conditions a similar leaf number per plant in WT
and transgenic lines. When subjected to drought, WT
kept the same leaf number but the leaves wilted
(Fig. 2a, b), however transgenic lines exhibited sig-
nificant decline in leaf number (0.05 ± 0.01,
0.13 ± 0.02 and 0.10 ± 0.02 % respectively for
WT, L15 and L20) (Table 6, Supplemental Table 6).
The distinction between WT and transgenic lines
was additionally confirmed through multivariate
analysis. The PCA-BGA plot (Fig. 4) showed that
all control plants were discriminated from corre-
sponding stressed plants along the 1st axis which
explain 60 % of variation. Along the second axis
which explained 20 % of variation it was possible to
discriminate stressed transgenic from stressed WT
plants. Among the tested parameters, seven out of
eleven were significantly correlated with the 1st
axis. These parameters include shoot and root DW,
root DW/FW ratio, RWC, WRA, gs and Amax.
While the 2nd axis was defined by shoot and root
DW/FW ratios and osmotic potential. It’s clear
through this PCA analyse that no dissimilarities
between control plants of both WT and transgenic
lines exist. Through PCA-BGA analysis (Fig. 5)
regarding only stressed plants, it was possible to
separate L15 from L20 with the contribution of
shoot DW/FW ratio.
4 Discussion
In this study, we investigated the effect of the
constitutive expression of the VviRD22a gene in
enhancing adaptation to drought in tobacco from
germination to adult plant stages. The VviRD22a gene
(accession No AY634282) was identified from grape-
vine and revealed to be induced in salt tolerant
genotypes (Hanana et al. 2008; Daldoul et al. 2010)
and related with increased salt tolerance in tobacco
(Jardak Jamoussi et al. 2014). The assessment of
VviRD22a gene for genetic engineering drought
tolerant plant would be of great interest for grapevine
breeding program. Gene candidate for breeding needs
to be identified and a screening under in vitro and
greenhouse conditions would be necessary to validate
the transgenic expression contribution toward drought
tolerance enhancement.
In this study, we evaluated the in vitro and ex vitro
physiological responses of VviRD22 expressing L15
and L20 transgenic tobacco lines to water stress.
Fig. 2 Wild type (WT) and L15 and L20 VviRD22a-expressing
tobacco plants under a control conditions and b after 9 days of
water stress
404 Theor. Exp. Plant Physiol. (2016) 28:395–413
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According to the in vitro assays, we noticed that under
control conditions (0 mM Mannitol; 0 % PEG),
transgenic seeds exhibited faster germination capac-
ities compared to WT. This would imply that
VviRD22a expression may confer a higher water
uptake capacity to transgenic seeds, making water
available to the embryo before metabolic activity
resumption (Mohr and Schopfer 1995). Additionally,
under Mannitol and PEG treatments, transgenic seeds
had higher germination abilities, compared to WT.
Comparison of these treatments revealed that PEG
was more discriminative between transgenics and WT
comparing to Mannitol. This would be related to
distinct lines sensibility to these stress inducers that
may involve distinct mechanisms. In both seed and
vegetative stages, Abe et al. (2003) showed that
transgenic Arabidopsis thaliana, over-expressing the
AtMYC2 and AtMYB2 genes controlling rd22 gene
expression (Abe et al. 1997), displayed an improved
osmotic stress tolerance due to significant hypersensi-
tivity to ABA and resulted in an up-regulation of the
rd22. Based on these reports, we suggest thatVviRD22a
constitutive expression contribute to water stress
tolerance at both germination and seedling stages. This
effect would involve a cellular protection of the seed
tissues and would allow subsequently a better cell
growth. Our results are in accordance with those of
Wang et al. (2012), who reported that ectopic expres-
sion of Glycin max rd22 in transgenic BY-2 cells could
significantly reduce the percentage of PEG induced cell
death, suggesting direct cellular protection ability for
GmRD22 under osmotic stress. This gene would
regulate cell wall peroxidase activity and hence cell
wall properties and integrity (Wang et al. 2012).
To investigate the in vivo role of VviRD22a in
drought tolerance improvement, we conducted a
drought assay under greenhouse conditions and eval-
uated fresh and dry biomasses, stomatal conductance
(gs), photosynthesis rate (Amax), RWC, WRA and
osmotic potential.
In terms of growth, shoot and root biomasses were
higher in the transgenic tobacco compared to WT,
despite the significant decrease in both under drought.
Wang et al. (2012) reported that Gmrd22 expression
could alleviate the negative effect of PEG on the root
elongation of transgenic Arabidopsis and improve the
Table 5 Comparison water status of WT and VviRD22a- expressing tobacco plants (lines L15 and L20) through RWC, WRA, Ws at
the end of the drought assay (day 9)
RWC WRA Ws
WT-C 92.67 ± 0.18a 84.05 ± 1.53a 1.06 ± 0.04a
L15-C 72.5 ± 1.44c 83.5 ± 4.25a 1.08 ± 0.002a
L20-C 82.31 ± 3.27b 82.31 ± 1.33ab 1.1 ± 0.06a
WT-ST 50 ± 2.62e 53.33 ± 3.33c 1.12 ± 0.05a
L15-ST 61.88 ± 1.05d 77.68 ± 0.09ab 1.33 ± 0.02b
L20-ST 68.83 ± 1.68c 75.65 ± 1.69b 1.34 ± 0.08b
One-way Anova
All genotypes
F-ratio 50.5326 22.4658 6.5863
F-critical 3.1059 3.1059 3.1059
WT 9 Stress
F-ratio[F-critical Yes Yes No
L15 9 Stress
F-ratio[F-critical Yes No Yes
L20 9 Stress
F-ratio[F-critical Yes Yes No
Data are mean ± SE of 3 replicates. One-way ANOVA was preformed to detect significant interactions between genotypes and
treatments. When found significant, the least significant differences (LSD) were calculated
The letters indicate significant differences according to LSD test (p B 0.05)
Theor. Exp. Plant Physiol. (2016) 28:395–413 405
123
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survival rate under drought. Moreover, it was reported
that under drought conditions, roots may produce
chemical signals, such as ABA, which can be trans-
ported to the shoots where an array of physiological
changes occurs to control water loss (Martin-Vertedor
and Dodd 2011). Such observation was also reported
under water stress and other abiotic constraints such as
salinity (Ghanem et al. 2011), temperature (Malik
et al. 2013) which are sensed by roots and may
influence hormone signaling between roots and
shoots. This would consequently induce changes in
processes controlling shoot physiology (Perez-Alfo-
cea et al. 2010, 2011).
Our results also showed that transgenic plants had a
specific stomatal behaviour based on a significant
decrease in conductance since the first three days of
water stress application and higher gs values than in
WT on later stages (6th and 9th days of drought). This
indicates an early stomatal control ensuring later a
physiological adjustment as was registered by Amax.
Such a behavior seems to be one of the bases of
VviRD22a-induced drought tolerance. According to
Turner et al. (1986), stomatal conductance has always
been considered as a favorable criterion for drought
adaptation and stomatal closure is one of the first and
main strategies for drought tolerance as it limits water
loss by transpiration (Tardieu 2003; Rambal et al.
2003; Sperry 2000; Wilkinson et al. 2001; Zhu 2002;
Chaves et al. 2003). Simultaneously to the stomatal
regulation a moderate reduction in the photosynthetic
rate was registered in the transgenic lines compared to
WT. It is well known that drought limits the
availability of CO2 and thus tends to inhibit photo-
synthesis (Cornic 2000; Chaves et al. 2003), since that
stomatal conductance decrease affects CO2 diffusion
under water stress (Flexas et al. 2002; Warren et al.
2004). Consequently, the more or less rapid stomatal
response results from a compromise between the
reduction of CO2 assimilation and the need to avoid
dehydration (Goh et al. 2003, 2009; Ludlow and
Muchow 1990). These authors suggested that the
induction of the specific RD22 gene in Arabidopsis
could have a significant role in controlling stomatal
movement in response to increased endogenous ABA
concentration. In this context, it has been demon-
strated that MYB transcription factors family plays a
Fig. 3 Stomata of WT (right panel) and L15 transgenic line
(left panel) under control and drought conditions, observed after
six days of water stress by SEM. Bar, 50 lm. Difference in
stomata shape was observed under control conditions. L15 line
clearly exhibited an elliptical stomata shape (ovoid shape)
compared to WT under control and stress conditions
406 Theor. Exp. Plant Physiol. (2016) 28:395–413
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Table
6Comparisonofbiomassandphotosynthetic
param
etersofWTandVviRD22a-expressingtobacco
plants(lines
L15andL20)at
theendofthedroughtassay(day
9)
ShootFM
RootFM
ShootDW
RootDW
ShootDW/FW
RootDW/FW
Leafnumber
gs
Amax
WT-C
43.27±
1.11a
7.83±
0.92a
4.9
±0.06a
1.37±
0.22a
0.11±
0.002
0.18±
0.05
27.33±
0.88a
0,49±
0,04
30,52±
3,18a
L15-C
41.77±
0.78a
7.33±
0.37a
4.47±
0.17ab
1.47±
0.18a
0.11±
0.003
0.2
±0.02
27.67±
0.33a
0,52±
0,09
33,52±
1,37a
L20-C
42.8
±0.79a
7.367±
0.52a
4.63±
0.37ab
1.5
±0.11a
0.11±
0.008
0.2
±0.01
26.33±
0.88a
0,53±
0,06
37,43±
1,82a
WT-ST
24.77±
0.47c
3±
0.25c
2.67±
0.23d
0.5
±0.06b
0.11±
0.007
0.17±
0.02
27.33±
1.45a
0,18±
0,03
7,51±
0,12f
L15-ST
34.73±
1.16b
5.57±
0.37b
3.83±
0.18bc
1.16±
0.09a
0.11±
0.002
0.21±
0.02
21.33±
0.33b
0,31±
0,03
12,12±
0,66e
L20-ST
34±
0.46b
4.83±
0.3
b3.47±
0.42cd
1.1
±0.15a
0.1
±0.01
0.23±
0.04
21.67±
0.33b
0,3
±0,05
12,04±
0,29e
One-way
Anova
Allgenotypes
F-ratio
72.9233
13.5914
9.6208
6.4645
0.3317
0.4734
11.3259
6,9716
64,3149
F-critical
3.1059
3.1059
3.1059
3.1059
3.1059
3.1059
3.1059
3,1059
3,1059
WT9
Stress
F-ratio[
F-critical
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
L159
Stress
F-ratio[
F-critical
Yes
Yes
No
No
Yes
Yes
No
No
Yes
L209
Stress
F-ratio[
F-critical
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Dataaremean±
SEof3replicates.One-way
ANOVA
was
preform
edto
detectsignificantinteractionsbetweengenotypes
andtreatm
ents.When
foundsignificant,theleast
significantdifferences(LSD)werecalculated
Thelettersindicatesignificantdifferencesaccordingto
LSD
test(p
B0.05)
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123
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role in the stomatal response and therefore in regula-
tion of photosynthetic and related metabolism under
environmental stresses (Cominelli et al. 2005; Gray
et al. 2005; Liang et al. 2005; Jung et al. 2008; Saibo
et al. 2009). Results related to stomata aperture were
also noted by Ding et al. (2009) in Arabidopsis plants
exhibiting tolerance to water stress after the over-
expression of the Myb15 transcription factor allowing
an up-regulation of RD22 gene. Moreover, Seo et al.
(2009) revealed that the Arabidopsis activation tagged
mutant in which the Myb96 is constitutively over-
expressed, exhibited an enhanced resistance to
drought, so that MYB96-mediated signals enhance
plant resistance to drought by reducing stomatal
opening. They indicated consequently that MYB96
specifically regulates stomatal opening and that
among stress genes, only RD22 expression was
elevated in Arabidopsis plants. The involvement of
RD22 in stomatal regulation was also evoked byWang
et al. (2012) who supposed that an initial induction
likely associated to a rapid initial stomatal closure
occurs in the leaves from salinity stress-induced gene
expression pattern of Gmrd22. These reports corrob-
orate our findings that revealed a link between
VviRD22a gene expression and stomatal movement,
making transgenic tobacco able to enhance a rapid
adjustment mechanism. Such stomatal adjustments
were also revealed in our case at an ultrastructural
level. In fact, SEM showed clear differences between
WT and transgenic lines under control and drought
conditions which would evoke the hypothesis of
VviRD22a gene involvement in stomatal control.
To examine the contributions of VviRD22a to water
status in transgenic tobacco, RWC of WT and
transgenic plants was measured after 9 days of water
stress application. RWC was reported to be a good
b
a
Axis 1 Axis 2FW_Shoot (g plant-1) ns nsFW_Root (g plant-1) ns nsDW_Shoot (g plante-1) p < 0.000 nsDW_Root (g plante-1) p < 0.000 nsShoot DW/FW ratio (g plante-1) ns 0.039Root DW/FW ratio (g plante-1) 0.004 0.001RWC_Leaf (%) p < 0.000 nsWRA (%) p < 0.000 nsgs (mol m–² s–1) p < 0.000 ns Amax (μmol m–² s–1) p < 0.000 nsΨs (MPa) ns 0.000
1st axis: 60%2nd axis: 20%
Fig. 4 PCA analysis of WT
and transgenic tobacco lines
(L15 and L20) development.
a Under control conditions
(C: continuous irrigation)
and at the day 9 of drought
(ST) during the greenhouse
assay involving shoot and
root DW (g plant-1); shoot
and root DW/FW ratios; gs
(mol m-2 s-1); Amax
(lmol m-2 s-1); leaf RWC
(%); WRA (%), osmotic
potential (MPa).
b Correlation matrix
(Pearsons product
correlation coefficient) with
the associated p value
between the principal
components and the
analysed variables
408 Theor. Exp. Plant Physiol. (2016) 28:395–413
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indicator of plant water status at any given time since it
closely reflects the balance between water supply and
transpiration rate (Sinclair et al. 1995; Cominelli et al.
2005). It was largely reported that a stable RWC leads
to greater turgor pressures enabling a better growth
(Kirkham et al. 1980; Clarke and McCraig 1982;
Carter and Patterson 1985; Schonfeld et al. 1988). In
our case, under control conditions, RWC was lower in
transgenics than in WT, but does not affect biomass.
This can be due to a regulation mechanism that would
be related to VviRD22a overexpression in transgenic
control plants. Phenotypically, after 9 days of water
stress, transgenic plants were turgid, whereas WT
plants exhibited a severe leaf wilting. Other physio-
logical indices such as WRA (Jia et al. 2008; Zhang
et al. 2011) were less affected by drought in the
transgenic lines, in contrast to WT.
PCA analyses of WT and transgenic lines beha-
viour at the end of the assay do not reveal distinctive-
ness between transgenics and WT control plants. This
would suggest that phenotype related to VviRD22a
expression is not visible under well-watered
conditions. Under severe stress, PCA plots confirm
the dissimilarity in drought tolerance capacity of WT
and transgenic lines. The strong phenotype related to
the great aptitude of L15 and L20 to cope water stress
would be attributed to the dry biomass, water status
and the photosynthetic parameters. As the difference
between L15 and L20 was not relevant under stress
conditions (significance was only observed at the
shoot DW/FW ratio), it seems that the phenotype
related to VvRD22a constitutive expression is likely
associated to the transgene presence independently
from the insertion manner.
Comparison of VviRD22a gene expression contri-
bution to salt (Jardak Jamoussi et al. 2014) and to
drought tolerance improvement was investigated. In
fact, under stress conditions (salinity and drought),
differences between WT and transgenics stressed to
control (ST/C) ratios were significant regarding DM
and RWC (data not shown). The absence of significant
differences between transgenic plants ratios tested
during both assays reveals that maintenance of veg-
etative biomass and water status was exhibited in
b Axis 1 Axis 2FW_Shoot (g plant-1) ns nsFW_Root (g plant-1) ns nsDW_Leaf (g plante-1) 0.029 nsDW_Shoot (g plante-1) 0.000 nsShoot DW/FW ratio (g plante-1) ns 0.000Root DW/FW ratio (g plante-1) 0.004 nsRWC_Leaf (%) p < 0.01 nsWRA (%) p < 0.000 nsgs p < 0.008 nsAmax p < 0.000 nsΨs (MPa) 0.02 ns
1st axis: 69%2nd axis: 21%
aFig. 5 a Multivariate data
analysis of water stressed
transgenic lines (Line L15
and L20) and WT plants to
water stress during 9 days
comprising shoot and root
DW (g plant-1); shoot and
root DW/FW ratios; gs
(mol m-2 s-1); Amax
(lmol m-2 s-1); leaf RWC
(%); WRA (%), osmotic
potential (MPa).
b Correlation matrix
(Pearsons product
correlation coefficient) with
the associated p value
between the principal
components and the
variables analysed
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similar manner under salinity and drought and so
VviRD22a constitutive expression effect would be
comparable under theses constraints. Also, under
drought, roots ST/C DM ratios of WT were signifi-
cantly lower than that of transgenics leading to the
involvement of root part in transgenic plants defense
against drought. VviRD22a expression in transgenics
would contribute to drought tolerance by enabling root
protection. This assessment of VviRD22a constitutive
expression effects towards salinity and drought lead to
that VviRD22a expression in transgenics is more like a
stabilizer and not improver of biomass under control
and stress conditions, which is in contrast with other
insertions as VviDhn (Jardak-Jamoussi et al. 2016).
Here, we also confirm the protective role of VviRD22a
constitutive expression by biomass and water status
maintenance under water stress, including root pro-
tection under drought. Matus et al. (2014) reported the
absence of VviRD22 genes expression in root. It seems
in our case that the strategy to maintain root biomass
under stress may result from the stomatal adjustment
that together would contribute cooperatively to
provide physiological balance in water content ensur-
ing an improved water stress tolerance in transgenics
compared to WT.
Taken together, our results show that in transgenic
tobacco lines expressing VviRD22a, the stomatal
movement driving the diffusion of CO2 into the
mesophyll and water vapor to the atmosphere was
controlled distinctly. We would suggest then, that
VviRD22a transgenic expression improved drought
tolerance in tobacco by contributing to the control of
water vapour and/or CO2 diffusion through stomata
and the consequent regulation of stomata movement.
This would consequently maintain photosynthesis
efficiency and the ability to grow and preserve root
and shoot biomass under water constraint. Overall, our
findings related to salt (Jardak Jamoussi et al. 2014)
and drought tolerance enhancement by VviRD22a
gene would lead to the mechanisms underlying
protection of transgenics exposed to these constraints.
In conclusion, the in vitro and ex vitro assessment
of genetic engineered tobacco plants constitutively
expressing the VviRD22a showed an improved
drought tolerance from germination to the adult plant
stage. Our findings revealed in the transgenic tobacco
a positive correlation between stomatal conductance,
photosynthesis, growth and water status, allowing
confirmation of a signal enhancement controlling
effective water retention in the transgenic lines. This
water retention ability would be attributed to an
efficient physiological adjustment due to the constitu-
tive expression of VviRD22a gene that would conse-
quently have a significant potential for biomass
improvement under water scarcity, a limiting factor
for plant production. The VviRD22a transgenic
expression would be then used to attain an important
goal in the breeding program of grapevine and other
crops that can be exploited in marginal arid zones
since engineering of stomatal responses to reduce
water loss is an attractive approach to enhance drought
tolerance in crops (Schroeder et al. 2001). At present,
biochemical analyses are being undertaken for better
exploring the shoot proteome of VviRD22a transgenic
lines to deeply understand the mechanisms involved in
drought tolerance enhancement.
Acknowledgments This research is undertaken in the
framework of bilateral scientific cooperation between Tunisia
(CBBC) and Portugal (ITQB).
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