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Molecular BiotechnologyPart B of Applied Biochemistry andBiotechnology ISSN 1073-6085 Mol BiotechnolDOI 10.1007/s12033-012-9567-y

The Xerophyta viscosa Aldose Reductase(ALDRXV4) Confers EnhancedDrought and Salinity Tolerance toTransgenic Tobacco Plants by ScavengingMethylglyoxal and Reducing theMembrane DamageDeepak Kumar, Preeti Singh, MohdAslam Yusuf, Chandrama PrakashUpadhyaya, Suchandra Deb Roy,Thomas Hohn & Neera Bhalla Sarin

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RESEARCH

The Xerophyta viscosa Aldose Reductase (ALDRXV4)Confers Enhanced Drought and Salinity Tolerance to TransgenicTobacco Plants by Scavenging Methylglyoxal and Reducingthe Membrane Damage

Deepak Kumar • Preeti Singh • Mohd Aslam Yusuf •

Chandrama Prakash Upadhyaya • Suchandra Deb Roy •

Thomas Hohn • Neera Bhalla Sarin

� Springer Science+Business Media, LLC 2012

Abstract We report the efficacy of an aldose reductase

(ALDRXV4) enzyme from Xerophyta viscosa Baker in

enhancing the prospects of plant’s survival under abiotic

stress. Transgenic tobacco plants overexpressing ALD-

RXV4 cDNA showed alleviation of NaCl and mannitol-

induced abiotic stress. The transgenic plants survived

longer periods of water deficiency and salinity stress and

exhibited improved recovery after rehydration as compared

to the wild type plants. The increased synthesis of aldose

reductase in transgenic plants correlated with reduced

methylglyoxal and malondialdehyde accumulation and an

elevated level of sorbitol under stress conditions. In addi-

tion, the transgenic lines showed better photosynthetic

efficiency, less electrolyte damage, greater water retention,

higher proline accumulation, and favorable ionic balance

under stress conditions. Together, these findings suggest

the potential of engineering aldose reductase levels for

better performance of crop plants growing under drought

and salt stress conditions.

Keywords Abiotic stress � Aldose reductase �Methylglyoxal � Transgenics � Xerophyta viscosa

Abbreviations

FW Fresh weight

MDA Malondialdehyde

MG Methylglyoxal

ROS Reactive oxygen species

RWC Relative water content

TW Turgid weight

WT Wild type

Introduction

Different environmental stresses imposed on plants may

result in similar responses at the cellular and molecular

levels. This is due to the fact that diverse environmental

stresses often activate similar cell signaling pathways and

cellular processes, such as the production of stress proteins,

up-regulation of antioxidants, and accumulation of com-

patible solutes [1, 2]. It has been demonstrated that the

level of methylglyoxal (MG), a cytotoxic compound

increases upon exposure of plants to various abiotic stres-

ses [3] as does the content of reactive oxygen species

(ROS) [4]. At higher concentrations, MG is harmful to the

system as it is a potent cytotoxin and reacts with the major

macromolecules like RNA, DNA, and proteins leading to

cell death [5]. MG is mainly catabolized by two major

enzymatic pathways—the glyoxalase pathway [6] and an

alternate pathway involving aldose/aldehyde reductases

(ALR) that convert MG into acetol in an NADPH-depen-

dent two-step reaction [7].

Deepak Kumar and Preeti Singh contributed equally to this study.

D. Kumar � P. Singh � M. A. Yusuf � C. P. Upadhyaya �S. D. Roy � N. B. Sarin (&)

School of Life Sciences, Jawaharlal Nehru University,

New Delhi 110067, India

e-mail: neerasarin@rediffmail.com

Present Address:C. P. Upadhyaya

Department of Botany, Guru Ghasidas Central University,

Bilaspur 495009, India

T. Hohn

Institute of Botany, University of Basel,

Schonbeinstrasse 6, 4056 Basel, Switzerland

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DOI 10.1007/s12033-012-9567-y

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ALR (EC.1.1.1.21) belongs to the well-conserved aldo–

keto reductase super family of enzymes in plants and ani-

mals. They are monomeric, cytosolic proteins that catalyze

the NADPH-dependent reduction of a variety of carbonyl

metabolites including the aldehyde form of glucose which

is subsequently reduced to the corresponding sugar alcohol,

sorbitol, that is associated with maintenance of osmotic

balance of the cytoplasm and protection of macromolecules

in both animals and plant systems, under stress [8, 9].

The aldose reductase homologs have been shown to

have osmoprotective function and linked to the acquisition

of desiccation and freezing tolerance, as well as mainte-

nance of seed dormancy and longevity [10, 11]. The

recently characterized rice aldo–keto reductase (OsAKR1)

protected transgenic tobacco plants against oxidative and

heat stress [12]. An aldose reductase (ALDRXV4) gene was

cloned from the desiccation-tolerant plant Xerophyta vis-

cosa Baker, and identified because it was able to confer

tolerance to severe water loss in a desiccation-intolerant

mutant Escherichia coli strain [13].

In this study, we functionally validated the efficacy of

overexpression of ALDRXV4 gene in the model plant

tobacco in enhancing the tolerance of transgenic plants to

drought and salinity stress as compared to wild type (WT)

plants. We also investigated the plausible mechanism for the

protection and found that increased detoxification of MG,

decreased lipid peroxidation, and an increase in the content

of osmolytes—sorbitol and proline contribute to the

enhanced tolerance of these transgenic plants against stress.

Materials and Methods

Vector Construction and Tobacco Transformation

The full-length cDNA of ALDRXV4 (accession no.

AF133841) cloned under CaMV 35S promoter in pGEMT

easy cloning vector (Promega, WI, USA) was provided by

Prof. Jennifer A. Thomson (University of Cape Town, South

Africa). The cDNA with CaMV 35S promoter was amplified

with primers containing NheI and BstEII restriction sites

nested within the forward (50AAAGGCTAGCGTTGAAG

ATGCCTCTGCCGAC30) and reverse (50GTAAGGTGACC

TTGCAATTACTTTAGACTTCACCG-30) primers, respec-

tively. The binary vector pCAMBIA 2301 (CAMBIA,

Canberra, Australia) used in this study contains a 2.8 kb

GUS expression cassette and neomycin phosphotransferase

(nptII) gene driven by the CaMV 35S promoter. The GUS

gene along with its upstream CaMV 35S promoter and lacZ awas removed by double digestion with restriction enzymes

XbaI (site present in the pUC18 MCS of pC2301) and BstEII

(site present upstream of the nos poly A in pC2301). The

PCR-amplified CaMV 35S-ALDRXV4 cDNA described

above was then cloned in this vector between the XbaI and

BstEII sites. The NheI and XbaI sites were, however, not

restored after this ligation. The construct, thus formed, was

named pCAM-ALDRXV4. It contained the neomycin phos-

photransferase gene (nptII) as the plant selectable marker.

The construct was mobilized into Agrobacterium tumefac-

iens strain GV3101 by freeze–thaw method [14]. Agrobac-

terium-mediated transformation of tobacco (Nicotiana

tabacum cv. Xanthium) leaf disks was performed according

to the standard protocol of Horsch et al. [15]. Regenerated

shoots were separated from the calli and transferred onto

Murashige and Skoog’s (MS) medium, with 100 mg/l

kanamycin for selection of the transformants. The plantlets

were subsequently hardened for 2 weeks and transferred to

soil for flower and seed setting.

Genomic DNA PCR and Southern Blot Analysis

Total genomic DNA was extracted from leaves of the

putative transformed and WT plants using the CTAB

method [16] and quantitated spectrophotometrically. The

presence of the transgene (ALDRXV4) in the T0 transgenic

lines was confirmed by PCR analysis using the same

primers as were used in the cloning of ALDRXV4 cDNA.

The amplified products were separated by electrophoresis

on a 0.8 % agarose gel.

Southern blot analysis was performed according to

standard procedures [17]. Genomic DNA (10 lg/lane)

from WT and PCR positive plants was digested with BstEII

and separated by electrophoresis on a 0.8 % agarose gel

and blotted onto a nylon membrane (Amersham Pharmacia,

USA). [a32P] dCTP-labeled probe (amplified from pCAM-

ALDRXV4 by PCR using the primers described above) was

prepared using Random Primer Labeling Kit (Amersham

Pharmacia Biotech, UK) and used for hybridization of the

membrane at 65 �C using standard procedures [17].

RT-PCR Analysis

Total RNA was isolated from leaves of both transgenic and

WT plants using TRIzol reagent (Invitrogen, CA, USA)

and treated with RNase-free DNase I in order to remove

any contaminating genomic DNA. RT-PCR amplification

was conducted using an RT-PCR kit (AccuScript, Strata-

gene, USA) in accordance with the manufacturer’s

instructions. Total RNA (1 lg) was used for generation of

first-strand cDNA using MMLV reverse transcriptase. The

gene specific primers for amplification of ALDRXV4 cDNA

were 50-AAAGGCTAGCGTTGAAGATGCCTCTGCCGA

C-30 and 50-GTAAGGTGACCTTGCAATTACTTTAGAC

TTCACCG-30. The primers for tobacco actin (as internal

control) were 50-AGTAAGGTGACCTTGCAATTACTTT

AGACTTCACCG-30 and 50-AAAGGCTAGCGTTGAAG

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ATGCCTCTGCCGAC-30. The RT-PCR products were

visualized by electrophoresis on 1.2 % agarose gel.

Western Blot Analysis

To check the expression levels of ALDRXV4, soluble protein

was extracted from transgenic and WT tobacco plants. Young

leaves (100 mg fresh weight [FW] from transgenic as well as

untransformed control plants were ground in 200 ll extrac-

tion buffer (20 % glycerol, 62.5 mM Tris–HCl, pH 6.8, 5 %

2-mercaptoethanol, and 0.1 % SDS). The extract was centri-

fuged at 13,000 rpm for 20 min at 4 �C and the supernatant

was used for ALR enzyme assays and for western blot anal-

ysis. Protein concentration was estimated by the method of

Bradford [18]. Protein extracts were fractionated using 12 %

SDS-PAGE and transferred to nitrocellulose membrane

(Sigma-Aldrich) using the semi-dry transfer system (Owl

Separation Systems, UK). Transfer of proteins onto the

membrane was checked by staining with Ponceau S stain [19].

The membrane was briefly rinsed in Tris Buffered Saline with

Tween-20 (TBST) and incubated in blocking solution (2 %

BSA in TBST) for 1 h with gentle shaking at room tempera-

ture. The membrane was given brief washes (5 min each)

thrice with TBST and incubated for 1 h with maize aldose

reductase rabbit antiserum (1:2,000 dilution). The membrane

was washed thrice with TBST (5 min/wash), after which it

was incubated with secondary goat anti-rabbit IgG, conju-

gated with horse-radish peroxidase (Sigma-Aldrich; 1:5,000

dilution), for 1 h. The membrane was incubated with diam-

inobenzidine (DAB) prepared in TBS, till the color developed.

All the dilutions of primary and secondary antibodies were

made in TBST containing 0.3 % BSA.

Seed Germination and Seedling Growth

Seed germination was studied in three sets of *100 seeds

each of WT and T1 progeny of the transgenic lines. The

seeds were surface sterilized and placed on MS agar plates

with or without mannitol (200 mM) and NaCl (200 mM),

incubated at 25 ± 2 �C under 16 h light/8 h dark photo-

period with the light intensity of 50 lmol/m s-1. Germi-

nation was scored daily and percentage germination was

calculated after 7 days.

T1 seedlings were also tested for growth on different mannitol

and NaCl concentrations. The 7-day-old WT and kanamycin-

positive T1 seedlings were transferred to MS medium supple-

mented with 100, 200, and 300 mM mannitol or 100, 200, and

300 mM NaCl. Growth was visually followed up to day 15.

Leaf Disk Senescence Assay

Healthy and fully expanded leaves from the transgenic as

well as WT plants were briefly washed with sterile distilled

water and leaf disks of 1 cm diameter were punched out

and floated in a 10 ml solution of mannitol (200, 400, 600,

and 800 mM, 3–4 days), NaCl (200, 400, 600, and

800 mM, 3–4 days), or MG (5 and 10 mM, 2 days),

respectively. In all the above treatments, leaf disks floated

on distilled water served as experimental control. The

chlorophyll content in the leaf was estimated according to

the procedure of Arnon [20].

Stress Tolerance Experiments

For drought and salt stress treatments 6-day-old seedlings of

WT grown on MS plates and transgenic lines grown on MS

plates supplemented with 100 mg/l kanamycin were trans-

ferred into plastic pots containing agropeat and vermiculite

(3:1 v/v) in a growth chamber at 25 �C, 60 % relative

humidity, and 16 h light/8 h dark photoperiod with light

supplied at an intensity of 150 lmol/m2 s-1. For drought

stress WT and transgenic plants were left unwatered for

14 days and then rewatered for 6 days for recovery.

For salt stress treatment, the plants were irrigated with a

200 mM NaCl solution for 14 days and then with water for

6 days. For tolerance of the plants under abiotic stress

throughout their life cycle, seedlings selected on kanamycin

were transferred to earthen pots and grown in a glasshouse

(16 h light/8 h dark, 25 ± 2 �C). Starting 2 weeks after

transfer, the plants were watered biweekly with mannitol

(200 mM) or NaCl (200 mM) solution or water. These plants

were used for performing different experiments.

Measurements of Chlorophyll Fluorescence

Photosynthetic activity was measured as photochemical

yield (Fv/Fm), which represents the maximum quantum

yield of photosystem II, by recording the chlorophyll

fluorescence using a portable chlorophyll fluorescence

meter (Handy-PEA, Hansatech, UK) after 30 min of dark

adaptation of the leaves. Measurements were conducted at

room temperature (25 �C) using saturating light pulse of

white light (8,000 lmol/m2 s-1 for 0.8 s).

Measurement of relative water content (RWC)

For determination of RWC, fresh leaves were detached and

weighed immediately to record FW, followed by dipping

them in distilled water for 12 h. The leaves were then

blotted to wipe off excess water, weighed to record turgid

weight (TW), and subjected to oven drying at 70 �C for

24 h to record dry weight (DW). The RWC was determined

by the equation:

RWC ¼ ðFW� DWÞ � 100=ðTW� DWÞ:

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Measurement of Electrolyte Leakage

Electrolyte leakage was determined according to the

method described by Sairam and Srivastava [21]. Leaves

were collected and washed with deionized water for three

times in order to remove surface-adhered electrolytes. They

were then placed in test tubes and immersed in 10 ml of

deionized water and the electrical conductivity (EC1) was

measured. After incubation at 55 �C for 30 min the EC2

was determined again. The samples were heated in boiling

water for 1 h before the total conductivity was measured in

the solution (EC3). Relative ion leakage was expressed as a

percentage of the total conductivity:

Relative Electrolyte Leakage¼ ½ðEC2�EC1Þ=EC3� � 100:

Measurement of Aldose Reductase Activity

The ALR enzyme activity was determined by monitoring

the decrease in OD at 340 nm due to NADPH utilization

[22]. The reaction mixture made in 0.1 M potassium

phosphate buffer (pH 6.2) contained 0.15 mM of NADPH,

20 mM MG as substrate, and the crude protein extract in a

final volume of 1 ml. The reference cuvette (blank) con-

tained everything except the substrate.

Estimation of Sorbitol

Sugar alcohols were estimated as described by Pommerr-

enig et al. [23]. Sugar alcohols were extracted from the

frozen powdered material by incubation in 80 % ethanol at

80 �C for 4 h. After centrifugation at 2,00,0009g for

15 min, 800 ll of the ethanolic supernatant was transferred

into a new tube and the ethanol was completely evaporated

under vacuum. The dry residue was dissolved in distilled

water, and the sorbitol content was determined enzymati-

cally with the use of a D-sorbitol colorimetric assay kit

(BioVision Inc., CA, USA). D-Sorbitol was oxidized by

NAD?-dependent sorbitol dehydrogenase to D-fructose and

the reduced NADH was consumed in a diaphorase reaction

for the reduction of iodonitrotetrazolium. The absorbance

of formazan product, thus formed, was measured at 560 nm

and the sorbitol content was estimated as per the manu-

facturer’s instructions.

Estimation of Methylglyoxal

MG was estimated according to the method of Yadav et al.

[3]. About 0.5 g leaf tissue was homogenized in 3 ml of

0.5 M perchloric acid. After incubating for 15 min on ice,

the mixture was centrifuged at 4 �C for 10 min at

11,0009g. The supernatant was decolorized by adding

charcoal (10 mg/ml), kept for 15 min at room temperature,

and centrifuged at 11,0009g for 10 min. Before using this

supernatant for MG assay, it was neutralized by incubating

with saturated solution of potassium carbonate for 15 min

at room temperature and centrifugation at 11,0009g for

10 min. The neutralized supernatant was used for MG

assay. In a total volume of 1 ml, 250 ll of 7.2 mM 1,2-

diaminobenzene, 100 ll of 5 M perchloric acid, and 650 ll

of the neutralized supernatant were added in that order. The

absorbance at 335 nm of the derivatized MG was read after

25 min in a Hitachi U-2000 spectrophotometer (Hitachi,

Japan). The final concentration of MG was calculated from

the standard curve and expressed in terms of lmol/g FW.

Measurement of MDA Content

The tobacco leaves were ground to a fine powder under

liquid nitrogen. 3 ml of 10 % trichloroacetic acid was

added to 0.2 g of the powder and left at 4 �C overnight.

After centrifugation at 1,0009g for 20 min, the supernatant

was transferred to a new tube for measurements. 2 ml of

0.6 % thiobarbituric acid (TBA) was added to 2 ml of the

supernatant. The mixture was vortexed thoroughly, heated

in boiling water for 15 min, cooled immediately and cen-

trifuged. Absorbance values of the supernatant were

detected at wavelengths of 532 and 450 nm using water as

blank. The formula for the calculation of MDA content

was:

MDA content (lmol/l) ¼ 6:45� OD532 � 0:56� OD450

Estimation of Proline

Proline content was estimated using the standard protocol

[24]. 100 mg of leaf tissue was crushed in liquid N2 and

homogenized in 10 ml of 3 % sulfosalicylic acid. The

homogenate was centrifuged at 15,000 rpm and to 2 ml of

the upper aqueous phase, 2 ml of 0.2 % ninhydrin and 2 ml

of glacial acetic acid were added. After incubation at

100 �C for 1 h, reaction was terminated on ice. Proline was

extracted from the mixture with 4 ml of toluene. The

optical density of the upper aqueous phase was measured at

520 nm and proline concentration was determined from the

standard graph made using the purified L-proline (Sigma,

MO, USA) and calculated on a FW basis (lmol/g).

Estimation of Endogenous Ion Content

For determination of endogenous Na? and K? contents,

100 mg leaf tissue (unstressed and salt-treated) of WT and

transgenic plants was taken and digested in 0.1 % HNO3.

Ions were extracted in distilled water by boiling for 30 min

twice. The filtered extract, thus obtained, was used to

measure specific ions using a flame photometer.

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Statistical Analysis

All experiments were performed at least three times inde-

pendently. Results were assessed by Student’s t test. Sig-

nificance was defined as P \ 0.05.

Results

Generation and Analysis of Transgenic Tobacco Plants

Overexpressing ALDRXV4 cDNA

Six different tobacco transgenic lines (X1, X2, X3, X4, X5,

and X6) were generated by overexpressing ALDRXV4 gene

under the control of constitutive CaMV 35S promoter using

the construct shown in Fig. 1a. Integration of the ALD-

RXV4 transgene into T0 transgenic tobacco plants was

confirmed by PCR using the gene specific primers (data not

shown) and Southern blot analysis using CaMV 35S-

ALDRXV4 specific fragment as probe (Fig. 1a). While a

single-copy transgene insertion was seen in the lines X1,

X2, X3, X4, and X6, two-copies of the transgene were

found to be integrated into the genome of the transgenic

line X5 (Fig. 1b). The RT-PCR analysis showed that

mRNA of ALDRXV4 was successfully expressed in the six

transgenic lines (Fig. 1c), whereas it was absent in the WT

plant. Western blot analysis of representative lines using

polyclonal antibodies raised against maize ALR showed

the presence of a single prominent band of expected size

(36 kDa) confirming the expression of the transgene at

protein level (Fig. 1d). The transgenic plants grew nor-

mally in glass house and showed no morphological dif-

ferences when compared to the WT plants, thus, indicating

that expression of ALDRXV4 had no deleterious effects on

the morphological growth of these plants.

Overexpression of ALDRXV4 Enhances Drought

and Salt Stress Tolerance During Germination

and Early Seedling Development

To study the effect of overproduction of ALDRXV4 on the

germination of transgenic tobacco seeds exposed to

drought and salt stress, seeds of WT and T1 transgenic lines

(X1–X6) were germinated on MS medium (without kana-

mycin selection), supplemented with 200 mM mannitol

and 200 mM NaCl to simulate drought and salt stress,

respectively. The germination rates of WT and transgenic

seeds under non-stressed (MS medium) conditions were

similar and no phenotypic difference was observed in the

developing seedlings. However, germination of WT seeds

was significantly impaired under stress conditions when

compared to transgenic seeds. The germination of WT

seeds on medium supplemented with 200 mM mannitol

and 200 mM NaCl was 40 and 38 %, respectively, whereas

WT X1 X2 X3 X4 X5 X6B

ALDRXV4

CWT X1 X2 X3 X4 X5 X6

Actin

DWT X1 X2 X3 X4 X5 X6

36 KDa

Ponceau

A

probe ~ 1.3 kb

35ST 35SP lox

RBLB

lox 35SPnptII nosTXV4

BstEII

Fig. 1 Transformation of tobacco plants for overexpression of

ALDRXV4. a Schematic representation of the pCAM-ALDRXV4construct used for tobacco transformation. The BstEII restriction site

used in the Southern analysis is marked. The region for amplification

of the probe used in the Southern analysis is also marked. b Southern

blot analysis showing the integration and copy number of the

transgene. c RT-PCR analysis confirming expression of ALDRXV4 in

young fully expanded leaves of transgenic tobacco plants. Actin gene

was used as an internal control. d Western blot analysis of total

protein extract from leaves of WT and transgenic tobacco lines.

Lower panel shows the Ponceau stained membrane after transfer for

checking the equal loading of proteins. WT tobacco control. (X1, X2,

X3, X4, X5, and X6) independently transformed T1 transgenic lines

of tobacco

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the seeds of the transgenic line X3 exhibited the best

response with a germination percentage of 90 and 85,

respectively, under these conditions (Fig. 2a, b). This line

along with two other transgenic lines, X1 and X2, were

chosen for further experimentation.

In order to study the effect of abiotic stress on seedling

growth, 7-day-old seedlings of WT and kanamycin selected

transgenic lines grown on MS medium were transferred to

medium supplemented with 100, 200, and 300 mM man-

nitol and NaCl, respectively. The representative pictures

for the WT and transgenic line X3 are shown in Fig. 2c–h.

In the presence of mannitol, both WT and transgenic

seedlings showed growth retardation in a concentration-

dependent manner, but the retardation was comparatively

more in WT seedlings. The differences were not apparent

at 100 mM mannitol (Fig. 2c) but on increasing the con-

centration of mannitol to 200 mM, the seedlings of line X3

appeared green and healthy, whereas WT seedlings showed

severe growth retardation (Fig. 2d). At 300 mM mannitol,

the WT seedlings were severely affected and showed

chlorosis, while the transgenic seedlings continued to grow

(Fig. 2e). Similarly, when the seedlings were exposed to

100 mM NaCl, both WT and transgenic seedlings were

able to survive, but the WT seedlings were comparatively

smaller in size (Fig. 2f). At 200 mM NaCl, WT seedlings

were highly stunted in growth (Fig. 2g), whereas at

300 mM WT seedlings perished (Fig. 2h). However, the

transgenic seedlings survived at 300 mM salt concentration

(Fig. 2h).

ALDRXV4 Transgenic Tobacco Plants Tolerate

Mannitol and Salinity Stress and Resist Toxic Levels

of MG

Leaf Disk Senescence Assay

The response of transgenic lines to stress was tested using

the detached leaf disk senescence assay [25]. Leaf disks

from WT and ALDRXV4 transgenic plants were floated on

different concentrations of mannitol and NaCl for 3–4 days

or MG for 2 days. Representative pictures for the visual

changes in leaf disks of WT and highest overexpressing

200 mM Mannitol

0

20

40

60

80

100

WT X1 X2 X3 X4 X5 X6

% G

erm

iant

ion

A

0

20

40

60

80

100

WT X1 X2 X3 X4 X5 X6

% G

erm

inat

ion

200 mM NaCl

B

C D E

F G H

X3

WT

X3

WT

Mannitol

100 200 300 (mM)

NaCl

Fig. 2 Germination of seeds and growth of seedlings under mannitol

and salt stress conditions. Percentage germination of seeds from WT

and transgenic lines grown on MS medium supplemented with

200 mM mannitol (a) or 200 mM NaCl (b). Germination was scored

after 7 days. WT tobacco control. (X1, X2, X3, X4, X5, and X6)

independently transformed T1 transgenic lines of tobacco. The values

are mean ± SD from three independent experiments. Representative

phenotype of seedling growth on 100 (c), 200 (d), and 300 mM

(e) mannitol and 100 (f), 200 (g), and 300 mM (h) NaCl after 15 days

of treatment

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line X3, observed under different treatments, are shown in

Fig. 3a–c (lower panel). Quantitative measurement of the

chlorophyll content in these leaf disks is shown in Fig. 3a–

c (upper panel). The leaf disks from non-stressed WT

plants had comparable chlorophyll content to those from

the transgenic lines. However, chlorophyll content in the

leaf disks from transgenic plants was higher than WT by

1.1-, 1.2-, 1.3-, and 2-fold (line X1), by 1.1-, 1.1-, 1.2-, and

1.6-fold (line X2), and by 1.1-, 1.2-, 1.3-, and 2.3-fold (line

X3) at 200, 400, 600, and 800 mM mannitol, respectively

(Fig. 3a) On 200, 400, 600, and 800 mM NaCl solution the

chlorophyll content in the leaf disks from the transgenic

plants was higher than WT by 1.1-, 1.1-, 1.5-, and 3.8-fold

(line X1), by 1.1-, 1.1-, 1.3-, and 2.5-fold (line X2), and by

1.1-, 1.1-, 1.7-, and 4.5-fold (line X3; Fig. 3b). On

exposing the leaf disks to 5 and 10 mM MG, chlorophyll

content in the former case was 1.8-, 1.5-, and 2-fold higher

in the transgenic lines X1, X2, and X3, respectively, as

compared to the WT, whereas in the latter treatment it was

8.5-, 6.4-, and 10-fold higher in transgenic lines X1, X2,

and X3 compared to the WT plants (Fig. 3c). (Note that in

Fig. 3, data for only the highest overexpressing transgenic

line, X3, has been shown).

Stress Tolerance of Plants

In addition to the leaf disk senescence assays, the assess-

ment of the transgenic plants for tolerance towards drought

and salinity stress throughout their life cycle at the whole

plant level was also carried out. For testing the drought

tolerance of transgenic vs. WT plants, 6-week-old plants

were subjected to drought stress by withholding the water

supply for 14 days (see ‘‘Materials and Methods’’). While

the WT plants were severely wilted, the transgenic plants

showed less wilting (Fig. 4a, upper panel). After 14 days

of withholding irrigation, the plants were rewatered and

grown for 6 days to allow their recovery. The transgenic

plants showed better recovery and resumed faster growth

when compared with the WT plants. The transgenic plants

maintained turgidity of leaves even under water stress

condition, whereas the WT plants showed negligible

recovery, exhibiting severe bleaching of the leaves leading

to their death (not shown).

To test whether ALDRXV4 expression could enhance

salt tolerance, 6-week-old plants were irrigated with Hoa-

gland’s medium supplemented with 200 mM NaCl and the

plant survival was monitored daily. After 14 days, all the

WT plants were severely affected under these conditions as

evidenced by their stunted growth and chlorophyll loss.

However, only a few leaves of transgenic plants bleached

and their stems were still erect (Fig. 4a, lower panel). Upon

removal of salt stress, the transgenic plants showed better

recovery while WT plant showed negligible recovery and

ultimately perished (not shown).

These results suggest that the transgenic plants acquired

enhanced tolerance to drought and salinity stress. In order

to check whether the enhanced expression of ALDRXV4

gene would allow the transgenic plants to grow, mature,

and set seeds under osmotic (mannitol) and salinity (NaCl)

stress, WT and transgenic plants with three replicates each

were distributed in three groups. One group was irrigated

with mannitol (200 mM), the second group with NaCl

(200 mM) solution, and the third with water till the plants

reached maturity. The growth of WT plants was severely

affected under osmotic and salinity stress as evidenced by

their stunted growth and ultimate death, whereas the

transgenic plants grew, flowered, and produced normal

viable seed. Several growth parameters, such as plant

WT

X3

0

20

40

60

80

100

120

0 200 400 600 800

Chl

orop

hyll

(µg/

g F

W)

WTX3

A

0 200 400 600 800

WTX3

B

0 5 10

WT

X3

C

NaCl (mM) Methylglyoxal (mM)Mannitol (mM)

Fig. 3 Leaf disk senescence

under stress as measured by

chlorophyll content.

Chlorophyll content (lg/g FW)

of leaf disks from WT and

transgenic (line X3) tobacco

plants, kept for 3 days at

different concentrations of

mannitol (a), NaCl (b), and MG

(c). The values are mean ± SD

from three independent

experiments. The photographs

of WT and line X3 leaf disks

under the corresponding

treatments are presented below

each plot

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height, FW of leaves, time required for flowering, and seed

weight, were scored (Table 1) in order to compare the

performance of transgenic lines under stress vs. the WT

plants grown in non-stress conditions.

Transgenic Plants Overexpressing ALDRXV4 have

Improved Photosynthetic Efficiency, Higher Relative

Water Content and Less Membrane Damage Under

Drought and Salinity Stress

The photosynthetic efficiency (Fv/Fm) of the transgenic vs.

the WT plants was determined using the fluorescence mea-

surements made with Handy-PEA (see ‘‘Materials and

Methods’’). It was observed that under non-stress conditions,

the transgenic plants had slightly higher photosynthetic

efficiency as compared to WT plants. After 14 days of

exposure of plants to drought and salt stress, the Fv/Fm ratio

of WT plants decreased by 53 and 44 %, respectively, while

in transgenic plants the corresponding reduction was only 26

and 31 % for line X1, 27 and 33 % for line X2, and 23 and

27 % for line X3 (Fig. 4b). These results clearly showed that

transgenic plants had more robust photosynthetic machinery.

To investigate whether the enhanced tolerance of the

transgenic plants corresponded with their increased ability

to hold water, the RWC of leaves under drought and salt

stress conditions was measured. The RWC in leaf tissues of

WT plants was reduced from 83 % under non-stress to

45 % in drought stress condition and 43 % in salt stress,

whereas the decrease in the transgenic plants was from a

control value of 85, 84, and 86 % (in lines X1, X2, and X3)

to 73, 70, and 75 % under drought stress and 77, 75, and

79 % under salt stress condition, respectively (Fig. 4c).

Drought

stress

Salt

stress

WT X3A

0

1

2

3

4

5

6

7

Water Drought Salt

MD

A c

onte

nt (

µM g

-1

FW

of

leaf

)WT X1 X2 X3

E

0102030405060708090

Water Drought Salt

Ele

ctro

lyte

leak

age

(%)

WT X1 X2 X3

D

0102030405060708090

100

Water Drought Salt

RW

C (

%)

WT X1 X2 X3C

00.10.20.30.40.50.60.70.80.9

1

Water Drought Salt

Fv/

Fm

WT X1 X2 X3

B

Fig. 4 Comparison of drought

and salt stress tolerance of

ALDRXV4 overexpressing

transgenic and WT plants grown

in soil. a Morphology of WT

and transgenic (representative

line X3) tobacco plants growing

under the simulated drought

(14 days without irrigation,

upper panel) and salt stress

(irrigated with 200 mM NaCl

for 14 days, lower panel)conditions. b PSII

photosynthetic efficiency (Fv/

Fm) in the leaves of tobacco

plants after 14 days of drought

and salt stress. c RWC in the

leaves of WT and transgenic

plants under drought and salt

treatment. d Electrolyte leakage

from tobacco leaf tissue as a

percentage of total electrolytes

in the leaves under 200 mM

NaCl stress. e MDA content in

the WT and transgenic plants

under drought and salinity

stress. WT tobacco control. (X1,

X2, and X3) independently

transformed T1 transgenic lines

of tobacco. Data represent the

mean values (±SD) of three

independent experiments

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The electrolyte leakage in the WT plants were 40, 35.7,

and 42.8 % more as compared to the transgenic plants

(lines X1, X2, and X3, respectively) under drought con-

ditions, whereas it was 33.3, 26.7, and 36 % more as

compared to the transgenic plants (lines X1, X2, and X3,

respectively) under salt stress treatments (Fig. 4d). The

membrane damage due to lipid peroxidation was assessed

by measuring the MDA level as described in the ‘‘Materials

and Methods’’ section. The MDA content in the WT plants

increased by 2.3-fold under drought condition and by 2.03-

fold under salt stress compared to that in the WT plants

grown under normal non-stress condition. However, the

increase in MDA content was only 1.3-, 1.42-, and 1.2-fold

under drought and 1.4-, 1.46-, and 1.28-fold under salt

stress in the transgenic lines X1, X2, and X3, respectively

(Fig. 4e).

Enhancement of Aldose Reductase Activity

in ALDRXV4 Overexpressing Transgenic Plants

Corresponds with Decreased Methylglyoxal

and Increased Sorbitol and Proline Content

To gain more insight into the role of ALDRXV4 in pro-

viding tolerance to drought and salt stress, ALR enzyme

activity was measured in mature leaves of WT and trans-

genic plants. The enzyme activity in the transgenic plants

was 3.5-, 3-, and 4-fold-higher (in lines X1, X2, and X3)

relative to the WT plants when grown under non-stress

condition. Under mannitol (200 mM) stress, a 2.6-, 2.3-,

and 2.8-fold increase in the ALR activity was detected in

the transgenic plants (lines X1, X2, and X3) as compared to

WT plants (Fig. 5a). Similar results were obtained under

salt stress (200 mM NaCl), where the transgenic plants

(lines X1, X2, and X3) showed 3.8-, 3.6-, and 3.9-fold

enhancement in activity when compared with the WT

plants under similar stress (Fig. 5a). The level of MG was

almost similar in WT and transgenic plants under non-

stress condition. However, in response to drought (200 mM

mannitol) and salt stress (200 mM NaCl), the WT plants

exhibited 97 and 14 % increase in MG concentration

respectively, whereas this increase was only 48 and 11 %

in transgenic line X1, 69 and 14 % in transgenic line X2,

and 29 and 3.6 % in transgenic line X3 (Fig. 5b). The

sorbitol content in the leaves of transgenic plants was

found to be 1.2-, 1.16-, and 1.2-fold higher (in lines X1,

X2, and X3) than in the WT plants under non-stress con-

ditions. Under drought and salt stress conditions, the sor-

bitol content increased in both transgenic and WT plants.

However, the increase in sorbitol content in the transgenic

lines X1, X2, and X3 as compared to the WT plants was

1.6-, 1.2-, and 1.8-fold higher under drought conditions and

1.2-, 1.1-, and 1.3-fold higher upon salt stress treatment

(Fig. 5c). The level of proline was also found to increase in

both transgenic as well as WT plants. However, the

increase in the transgenic lines X1, X2, and X3 was,

respectively, 1.9-, 1.8-, and 2-fold higher than the WT

under drought stress while it was 1.9-fold higher in X1 and

X2 and 2.1-fold higher in X3 in comparison to the WT

plants under salt stress (Fig. 5d).

Na? and K? Accumulation in Response to Salt Stress

The Na? and K? content in the leaves of WT and trans-

genic plants treated with 200 mM NaCl vs. the non-stress

Table 1 Comparison of various growth parameters and seed production of the WT and transgenic plants (lines X1, X2, and X3) grown in the

presence of water, 200 mM mannitol and 200 mM NaCl, respectively

Parameters WT X1 X2 X3

Water

Height 115 ± 4.32 116 ± 2.0 112 ± 2.51 114 ± 2.94

Foliar biomass (g)a 3.3 ± 0.49 3.5 ± 0.30 3.1 ± 0.35 3.6 ± 0.20

No. of days to flower 121 ± 4.96 119 ± 4.04 120 ± 4.5 117 ± 5.09

Seed weight per pod (mg) 160 ± 4.89 164 ± 3.21 161 ± 4.0 165 ± 5.09

200 mM Mannitol

Height No survival 105 ± 3.51 101 ± 2.51 110 ± 7.54

Foliar biomass (g)a – 2.5 ± 0.20 2.0 ± 0.30 3.1 ± 0.72

No. of days to flower – 133 ± 3.0 135 ± 3.51 132 ± 4.93

Seed weight per pod (mg) – 140 ± 4.04 132 ± 5.50 145 ± 6.55

200 mM NaCl

Height No survival 103 ± 3.0 100 ± 6.5 106 ± 3.85

Foliar biomass (g)a – 2.4 ± .30 1.9 ± .20 2.9 ± 0.29

No. of days to flower – 128 ± 2.6 125 ± 4.58 130 ± 3.74

Seed weight per pod (mg) – 135 ± 4.50 130 ± 5.68 138 ± 6.16

a The average weight of the fourth leaf from top. Each value is the mean of three replicates ±SD

Mol Biotechnol

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condition was measured. After 2 weeks of growth on

200 mM NaCl, the accumulation of sodium ions in the

leaves of WT plants was 1.8-, 1.6-, and 2-fold higher as

compared to that in the transgenic lines X1, X2, and X3

(Fig. 5e). The Na?/K? ratio of WT was 2 while transgenic

plants exhibited a ratio of 0.45, 0.55, and 0.3 (lines X1, X2,

and X3; Fig. 5f).

Discussion

Being sessile, plants cannot escape stress conditions

imposed on them by changes in their surroundings.

Therefore, in order to survive they have devised several

different mechanisms, through the evolutionary course, to

brace them against vagaries of the environment they grow

in. The study of extremophiles potentially offers new

insights into the adaptive mechanisms (and the genes

involved, therein) that such organisms employ while

growing under extremes of climate. X. viscosa, the ‘‘res-

urrection plant’’ that resuscitates and resumes normal

physiological activities after near-complete dehydration,

has been under investigation for mining of genes involved

in desiccation tolerance [26, 27]. Mundree et al. [13] iso-

lated an ALR (ALDRXV4) gene from X. viscosa plants. The

homologs of this gene have been identified from several

other plants and animals and their products have been

found to accumulate under different abiotic stress condi-

tions. The ALR catalyze the NADPH-dependent reduction

of carbonyl moieties. The accumulation of reactive car-

bonyls formed as a consequence of interaction of polyun-

saturated fatty acids of membrane lipids with the ROS

generated during the abiotic stress conditions is highly

deleterious to the cells. The role of ALR from several

organisms in combating these carbonyls and protecting

them from osmotic, salinity, drought, and UV stress has

been documented in the literature [12, 28–31].

In the present investigation, we evaluated the potential

of ALDXV4 in conferring tolerance to salinity and drought

stress in tobacco plants. Six independent transgenic events

were obtained following Agrobacterium-mediated trans-

formation and confirmed for transgene integration and

expression. Tobacco plants constitutively overexpressing

ALDRXV4 showed normal seed set in T0 transgenic lines.

There was no abnormal characteristic seen in the pheno-

type of the transgenic plants and in overall growth they

0

0.1

0.2

0.3

0.4

Water 200mM Mannitol 200mM NaCl

ALR

act

ivity

(U

/mg

prot

ein)

0

50

100

150

200

250

300

Water 200mM Mannitol 200mM NaCl

MG

(µM

/g F

W)

WT X1 X2 X3B

0

0.5

1

1.5

2

2.5

Water 200mM Mannitol 200mM NaCl

Sor

bito

l (nm

ol/µ

l) WT X1 X2 X3C

0

50

100

150

200

250

300

350

Water 200mM Mannitol 200mM NaClPro

line

cont

ent (

µ m

ol/g

DW

)

WT X1 X2 X3D

0

1

2

3

4

5

Water 200mM NaCl

mg/

g D

W

WT X1 X2 X3E

0

0.5

1

1.5

2

2.5

Water 200mM NaCl

Na+

/K+

ratio

WT X1 X2 X3F

A WT X1 X2 X3Fig. 5 Comparison of aldose

reductase activity, content of

some metabolites, and Na? and

K? ions in ALDRXV4overexpressing transgenic and

WT plants. Change in the level

of aldose reductase specific

activity (a), accumulation of

MG (b), sorbitol content (c),

and proline content (d) in

response to osmotic (200 mM

mannitol) or salinity (200 mM

NaCl) stress or water in the

leaves of WT and transgenic

plants. Na? content (e) Na?/K?

ratio (f) (percent DW of the

tissue) in leaf tissue of WT and

transgenic plants grown under

continued the presence of

200 mM NaCl or water. The

values are means (±SD) of

triplicate measurements

Mol Biotechnol

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were similar to WT plants growing under normal condi-

tions. The T1 transgenic plants showed increased tolerance

to mannitol and salinity (NaCl) stress as assessed by seed

germination and growth of the seedlings.

The tolerance was reflected not only at the germination

level but also during subsequent plant growth. After

14 days exposure to drought and salt stress, transgenic

plants grew normally whereas the WT plants perished.

Significant tolerance against stress conditions was also

observed in the leaf disk senescence assays. The leaf disks

from transgenic plants survived better during stress treat-

ments (mannitol, NaCl, and MG) by retaining higher

chlorophyll levels when compared to the WT, which

exhibited complete senescence of leaf disks due to reduced

level of chlorophyll. The transgenic plants showed a clear

advantage in overcoming the deleterious effect brought in

by MG toxicity in a concentration-dependent manner, thus,

implicating a role of ALDRXV4 overexpression in miti-

gating stress-induced damage to the photosynthetic appa-

ratus. MG is a glycolysis-derived toxic aldehyde and is a

known substrate for aldehyde reductases in organisms

including E. coli and yeasts [12, 32, 33]. High accumula-

tion of MG inhibits cell proliferation, degrades proteins by

modifying several amino acid residues and is, thus, toxic to

the cells [3, 34–36]. Glyoxalase pathway enzymes have

also been shown to detoxify MG in transgenic plants [36].

This signifies the existence of parallel pathways to bolster

the cellular defense against unwanted metabolites.

The damage caused by increased peroxidation of lipids

in the membrane bilayers as assessed by the MDA content

was found to be less in the transgenic tobacco plants

exposed to the two different stresses in comparison to the

WT plants. A pertinent consequence of the decreased MDA

levels was a better photosynthetic performance of the

transgenic plants compared to the WT plants. Accumula-

tion of MDA beyond a threshold level has been reported to

cause irreparable damage to the photosynthetic machinery

[37]. The ratio, Fv/Fm, is a measure of the proportion of

light absorbed by chlorophyll molecules associated with

PSII in the photochemical reaction and is, thus, an indi-

cation of actual photosynthetic efficiency [38]. The ability

of aldose reductase to modulate photosynthetic efficiency

has been demonstrated in tobacco plants expressing alfalfa

aldose reductase [30]. Under simulated abiotic stress con-

ditions (salinity and water stress) transgenic plants were

less sensitive to photoinhibition as compared to WT plants.

The build-up of various osmolytes, such as proline and

betaines has been positively correlated with stress tolerance

in many studies [39]. High proline content provides pro-

tection against ROS-induced disruption of photosystems

[40]. Proline is known to contribute to membrane stability

[39, 41] and has been widely documented as a parameter

for selection for salt stress tolerance. In this study,

significantly higher proline content was observed to accu-

mulate under stress conditions in the transgenic plants as

compared to WT plants. The mechanistic relation, if any,

between the ALR and proline content in the cells would,

however, require further experimentation.

The Na?/K? ratio was found to be relatively stable in

the leaves of the transgenic plants grown either under the

normal conditions or under drought and salt stress. This

revelation is in agreement with the generally accepted

understanding that the maintenance of Na?/K? homeosta-

sis is an important aspect of salt tolerance [42].

In conclusion, transgenic tobacco plants overexpressing

ALDRXV4 under the control of constitutive CaMV 35S

promoter have been generated. Our results, unequivocally,

establish the efficacy of aldose reductase gene from

X. viscosa in conferring tolerance against salt and drought

stress through detoxification of MG and reducing mem-

brane damage. Experiments are in progress to prove that

the results obtained in this study on a model plant translate

well for the crop plants.

Acknowledgments The authors thank Prof. Jennifer A. Thomson,

University of Cape Town, South Africa for ALDRXV4 cDNA and Dr.

Sylvia de Sousa, Universidade Federal de Sao Joao del-Rei, Brasil for

aldose reductase antibodies. Dr. Mikail Pooggin’s help in training

some members of NBS’ group during their visits to FMI and Uni-

versity of Basel, Switzerland, supported by Indo-Swiss Collaboration

in Biotechnology, is duly acknowledged. Dr. S.G. Mundree is

acknowledged for discussions related to this manuscript. DK and PS

are thankful to CSIR and UGC, India, respectively, for fellowships.

MAY is a UGC-Dr. D.S. Kothari Postdoctoral Fellow. This work was

funded by DST, India (Grant No. SR/SO/BB-37/2008). The research

in the lab of NBS is funded by grants from UGC-CAS, DST-FIST,

and DST-PURSE.

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