Author's personal copypinheiro/CP_PDFs/2016_TxPP_RD22.pdf · Donia Abdelwahed . Ne´jia Zoghlami . Asma Ben Salem . Olfa Zarrouk . Ahmed Mliki ... in pots ﬁlled with sandy soil

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

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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


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


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


123


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)


123


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)


123


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)


123


(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





123


(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


L20 x Stress






123


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


123


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





123


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


123


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)


123


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


123


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


123


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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Author's personal copypinheiro/CP_PDFs/2016_TxPP_RD22.pdf · Donia Abdelwahed . Ne´jia Zoghlami . Asma Ben Salem . Olfa Zarrouk . Ahmed Mliki ... in pots ﬁlled with sandy soil