Growth assessments of Nicotianatabaccum cv . Xanthitransformed with Arabidopsis thaliana P 5 CS under salt stress

1 Faculty of Basic Sciences, Department of Biochemistry, National Institute of Genetic Engineering and Biotechnology (NIGEB), P. O. BOX 14155 6343 Tehran, Iran. 2 Department of Plant Breeding and Agricultural Biotechnology, Faculty of Agriculture, Gorgan University of Agricultural Science and Natural Resources, Gorgan, I.R. Iran. 3 Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, Gatersleben, Germany. 4 Agronomy and Plant Breeding Department, College of Agriculture University of Tehran Karaj, 31587 77871, I.R. Iran.


INTRODUCTION
Drought and salinity are the two main limiting factors of plants growth and development.It is well known that application of selection programs that are effective in low stress conditions might lead to the selection of less tolerant cultivars (Perezde de vega, 1996).On the other hand, insensitive selection pressure could lead to the elimination of less adaptable genotypes, reduction of genetic (Paul et al., 1996;Sun et al., 1999).Accumulations of osmolytes in the cytoplasm stabilize cellular osmotic pressure which in turn reduces accumulation of toxic ions, enhances enzymes stability against toxic ions such as Na + , denaturing agents as well as osmotic stresses (Ashraf et al., 2007;Kishor et al., 2005;Okuma et al., 2000Okuma et al., , 2002)).
Among osmolytes, proline has shown to play a vital role against deleterious effects of ions and being involved in the stability of enzymes at higher temperatures (Banu et al., 2008;Arakawa et al., 1983;Arakawa et al., 1985;Schobert et al., 1978).Proline acts as a chemical chaperone conferring enzymes, conformational stability and activity (Ashraf et al., 2007;Okuma et al., 2000Okuma et al., , 2002;;Solomon et al., 1994).In addition, proline plays an important role in the adjustment of osmotic pressure of cells against stresses such as drought, salinity, low and high temperatures, insufficiency of nutrients, exposure to heavy metals, high acidity and ionizing radiation (Kishor et al., 2005).Parts of these activities could be attributed to the induction of the salt stress responsive genes, which possess proline responsive elements (PRE for example, ACTCAT) in their promoters (Chinnusamy et al., 2005).
The role of proline in osmo-adjustment has been reported in maize (Raya pati et al., 1991), "alfalfa" (Ginzberg et al., 1998) and Arabidopsis thaliana (Kiyosue et al., 1996).In maize, grown in low water potential, it was estimated that proline accumulation may account approximately 45% of the total osmotic adjustment in the root apex (Hasegawa et al., 2000;Votberg et al., 1991).Increased level of proline in response to osmotic stresses not only was observed in plants but also in a wide range of organisms such as bacteria, yeast, marine invertebrates and algae (Measues, 1975;McCue et al., 1990;Delauney et al., 1993).
Besides its stabilizing effects on cellular structure, proline is capable of detoxifying free radicals by forming long-lived adducts (Chen and Dickman, 2005;Chen et al., 2006;Floy et al., 1984;Okuma et al., 2004;Smirnoff et al., 1989).Hare and Cress (1997) hypothesized that, the NADP + / NADPH redox system may be important in metabolic regulation due to their involvement in the interconversion of the δ-1-pyrroline-5-carboxylate (P5C) to proline which could mediate the transfer of reducing equivalents from cytosolic NADPH to the electron transport (ET) chain.These inter-conversions could shuttle redox equivalents between cellular compartments, transfer of electrons from NADPH to NADP + , coupling the oxidation of NADPH to mitochondrial ET and serving as a mechanism for energy production (Phang, 1985).Yeh and Phang (1988) demonstrated that, P5C enhances the activity of the oxidative pentose phosphate pathway.
Accumulation of proline in response to many commonly encountered stresses in the natural environments has prompted suggestion that genetic engineering and thus, production of higher levels of proline in crops might play a benefactor role against environmental stress (Hare et al., 1997).Under abiotic stress, accumulation of proline is thought to result from the induction of δ-1-pyrroline-5carboxylate synthetase (P5CS) and pyrroline-5-caboxylate reductase (P5CR) which are involved in the biosynthesis of proline from L-glutamate (Ashraf et al., 2007).Also, inhibition of enzymes involved in oxidation of proline such as prolinedehyrogenase (PRODH) and pyrroline-5carboxylate dehydrogenase (P5CDH) would have positive impact on the proline accumulation (Delauney et al., 1993).
In Arabidopsis engineered with antisense proline dehydrogenase cDNA, the enzyme which is involved in the catabolism of proline, the results indicate accumulation of proline and constitutive tolerance to freezing temperatures as well as salinity (Nanjo et al., 2003).In accordance to mentioned findings, in the present study, Nicotianatabacum cv.Xanthi was transformed with the reverse transcribed P5CS cDNA of A. thaliana.The present report evaluates both morphological and physiological alterations in tobacco plants transformed with the A. thaliana P5CSin response to osmotic stress.

Complementary DNA (cDNA) synthesis
Total RNA was extracted from Arabidopsis seedlings using guanidine hydrochloride method (Clark, 1997) and treated with RNase-free DNase I. First strand cDNA was synthesized (expand reverse transcriptase, Roche) and the oligo dT18 primer as per recommendations of the supplier.After dilution, 50 to 100 ng of the resulting cDNA was used as template for polymerase chain reaction (PCR) amplification using primers: Forward; 5'-TCAGTTAACTGGATCCTCTCTCTGTGTGTGGTTTT-3' and Reverse; 5'-GAAGAGAAGCGAATTCAATGTTCTTCATCGTACTT-3', specific for the P5CS cDNA with two mismatches in order to create restriction sites for enzymes, BamHI and EcoRI (as underlined in primers sequences).The resulting product was cloned in BamHI / EcoRI sites of the plasmidpBluescript SK -(Stratagene) and transformed into Escherichia coli (DH5α) as a back up for P5CS for subsequent sub-cloning.Successful cloning was tested by applying restriction digestion, PCR amplification, dot blotting and sequencing.

Preparation of construct
In order to evaluate the effect of plant source of the original cDNA, P5CS from primary vector (pBluescript SK -/P5CS) was excised with XhoI and BamHI and used for subsequent subcloning and replacing the P5CS originated from Vignaaconitifolia (a kind gifted of Professor Verma, Ohio university, U.S.A. in pBI121 known as pBI P5CS (Kishor et al., 1995;Hong et al., 2000)) (Figure 1).The resultant P5CS was called pBI P5CS X, in which X stands for the applied XhoI restriction enzyme in the process of construction.This construct along with two other Vigna derived P5CS (Kishor et al., 1995;Hong et al., 2000) were used for Agrobacterium mediated tobacco plant transformation.All constructs were subsequently transformed into E. coli (strain DH5α) as back up and Agrobacterium tumefaciens for transformation.

Plant transformation and production of T 2 generation
Successful cloning, sub-cloning in vectors and transformation of plant were confirmed using restriction digestion, PCR, blotting, sequencing and reverse transcription polymerase chain reaction (RT-PCR).The Arabidopsis P5CS cDNA was sub-cloned into the binary vector; pBI121 as described earlier and used for plant transformation using Agrobacterium mediated transformation of tobacco leaf disks.Plants were also transformed with two Vigna P5CS cDNAs either with feedback inhibition or without inhibition as positive controls (Kishor et al., 1995;Hong et al., 2000).As an additional control (or control for the applied vector), pBI121 was also transformed to the plant leaf disks according to the earlier mentioned method.In addition, intact tobacco leaf disks were used as negative control.Following to callus formation, root and shoot forming plants were grown and after well adaptation of T 0 transgenic and control plants to the in vivo condition, they were transferred from in vitro culture medium to in vivo condition (pots containing vermiculite and composite; ½ : ½) and left for growth.Subsequently, plants were exposed to drought for confirmation of successful transformation in accordance with Yamchi et al. (2007).Transformed T 0 lines with high proline content were used for T 1 seed formation and plant production.T 1 seeds were cultivated in MS medium (Murashige et al., 1962)  ).Subsequently, plantlets were transferred into ½ vermiculite: ½ composite under earlier mentioned condition with 16 h of light and 8 h dark till grown well, proceeded self pollination and T 2 seed were produced.

Drought and salt treatment
T 2 generation of plants was treated with salt at both seed germination and growth phases.Twenty seeds of both T 2 and R 2 (wild type) obtained from the self pollinated T 1 and R 1 plants, were sown on MS-agar medium containing 0, 50, 100, 150, 200 and 250 mM NaCl.A 2 × 6 factorial experiment in a completely randomized design with 3 replicates was used.The experimental treatments included 2 plant types (T 2 transgenic seed versus R 2 wild type seed) at 6 levels of NaCl concentrations.After two weeks of sowing, percentage of germination was calculated on days 1, 3, 7 and 14. Analysis of variance on data was carried out using SAS version 6.12 software.Salt treatment of T 2 plants at growth phase was carried out by growing plants on 2 × 2 cm 2 plastic meshes placed on kanamycin-containing MS-agar medium.Three weeks later, plants were transferred into MS medium (suspension culture without agar with constant shaking for one month) containing 0, 50, 100, 200 and 250 mM NaCl.A 3 × 5 factorial experiment in a completely randomized design with 3 replicates was used.The experimental treatments were included 3 plant types (T 2 transgenic plant, R 2 wild type plant and plant transformed with pBI121) at 5 levels of NaCl concentration.
The plantlets were subsequently transferred and grown in pots as described earlier and seeds obtained from such plants were further cultivated in pots and used for evaluating drought and salt treatment of T 3 plants in vivo condition (Ferrario-Mery et al., 1998).For salt treatment, plants were exposed to ½ Hoagland's medium (Hoagland et al., 1938) containing 250 mM of NaCl for 21 days for salt stress and drought for 15 days.

Proline assay
Fresh and the uppermost well developed leaves (100 mg) of mature plant, were powdered in liquid nitrogen.Ten milliltres (10 ml) of 3% sulfosalicilic acid was added to each sample and centrifuged at 1300 rpm for 10 min.Two millitres (2 ml) of the supernatant from each sample and 2 ml of acidified ninhydrin solution (1.25 g ninhydrin dissolved in 30 ml acetic acid at boiling temperature to which 20 ml of ortho phosphoric acid was added) and 2 ml of 100% acetic acid were mixed and boiled for one hour.To stop further reaction, samples were immediately transferred onto ice-water for 20 min.To each samples, 4 ml of toluene was added at room temperature, mixed well and the absorbance at 520 nm was measured (Bates, 1973).For quantifying proline concentration, a calibration curve was prepared by measuring the absorbance of proline ranging in concentration from 1 to 160 µM.

Genomic DNA PCR and RT-PCR analysis
Based on the available cDNA sequence of the A. thaliana P5CS in NCBI (National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/,Acc No; D32138) two sets of primers were designed and genomic DNA PCR was carried out.In order to avoid non specific annealing of primers with endogenous tobacco P5CS, the forward primer was designed from the internal sequence of 35S promoter.As a result, amplification was restricted to plants with integrated exogenous P5CS (Figure 2a).The first of the first set of the primers were; 5 ′ -GGATTGATGTGATATCTCCACTGACG-3' which anneals to the internal sequence of 35S promoter as forward primer and 5 ′ -CCTTCAACATCGCTCAGAAGAATCAG-3' as the 5 ′ -GCAAGACTAAGTGGTAAAGTGGATCT-3 ′ , leading to 634 bp amplification product from 3' side of the P5CS.PCR mixture was composed of 1 µl DNA (25 to 50 ng), 10 pmole of each primer, 200 µM of each dNTP, 2 mM MgCl 2 , 1X PCR buffer and 1U Taq DNA polymerase (Roche) in a 25 µl final volume.PCR started with initial denaturation at 95°C for 5 min and followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 min and extension at 72°C for 1 min.Final extension was done at 72°C for 5 min.Post amplification product was electrophoresed in 1.2% agarose/tris-borateethylenediaminetetraacetic acid (TBE) gel at 80 V for 45 min and visualized with ethidium bromide staining.
In order to check transcription status of P5CS in transgenic, plant RT-PCR was carried out.Total RNA from transformants was isolated using RNeasyQiagen plant mini kit (Qiagen) and treated with RNase-free DNase I in order to validate the expression of exogenous P5CS and suspended in 30 µl of RNase-free water.cDNA was synthesized using M-MuLv reverse transcriptase (MBI, Fermentas).The RT-PCR was carried out using specific primer pairs for P5CS cDNA, such that, forward primer was composed of 5'-CCAAGGGCAAGTAAGATACTGAACAT-3' and the reverse primer 5'-GCAAGACTAAGTGGTAAAGTGGATCT -3', respectively.The amplification conditions were the same as that for genomic PCR and aliquot of each of the reactions were run on 1.2% agarose/TBE gel.

Southern blotting
Genomic DNA was isolated from young leaves of transgenic and wild type plants (Murray et al., 1980) and 30 µg of which were used along with 2 µg of each of the purified pBI-P5CS X plasmids as positive control and pBI121 plasmid as negative control in digestion reaction with Sac I and Bcl I restriction enzymes according to the restriction map of Arabidopsis P5CS cDNA.Digested DNA were resolved on a 0.7% agarose gel, blotted to nylon membrane (Roche), baked at 80°Cfor 2 h and prehybridized in a buffer containing 5X saline sodium citrate (SSC) (0.075 M NaCitrat, 0.75 M NaCl, pH7.0), 0.1% N-lauroylsarcosine, 0.02% SDS and 1% blocking reagent.Subsequently, overnight exposure to the denatured DIG labeled probe (Arabidopsis P5CS cDNA was ) decan)-4-yl) phenyl phosphate in detection buffer (0.1 M Tris-base, 0.1 M NaCl, 50 mM MgCl 2 pH 9.5) was added.Subsequently, the membrane was exposed to the x-ray film and kept in the dark till image development (Sambrook et al., 2001).

GUS assay
The GUS assay was performed according to Gallagher (1990).Leaf discs from control and transgenic plants were prepared and completely immersed in X-Gluc solution (0.1 M sodium phosphate buffer, pH 7.0; 10 mM EDTA; 0.5 mM potassizumferricyanide, pH 7.0; 1.0 mM Xglucuronide and 1% Triton X 100).Incubation was carried out at 37°C for at least 10 min or longer until formation of complete bluish-green color.

Peptide synthesis and production of anti Arabidopsis P5CS
The complete amino acid sequence of A. thaliana, ecotype; Columbia) was obtained from NCBI (Acc no; D32138, http://www.ncbi.nlm.nih.gov/) and 15 amino acids from N-terminal (EELDRSRAFARDVKR) was chosen for raising antibody against P5CS according to Nanjo et al. (1999).Polypeptide was synthesized (Sigma-Genosys) and the primary immunization was carried out by sub-coetaneous injection of 200 µg of polypeptide in complete Freund's adjuvant into 4 sites on rabbit back near the lymph nodes.Further boosting was done three times (intra-muscular) each at 4 weeks intervals with 200 µg of polypeptide prepared in the incomplete Freund's adjuvant (BioGene).After heart blood collection at the end of immunization, serum was collected and used for protein detection at 1/1000 dilution.

Immunoblotting
For immunoblotting, 200 mg fresh leaves from T 2 transgenic and control plants were powdered in liquid nitrogen, transferred into 1.5 ml of homogenizing buffer containing 10 mMTris-Cl pH 7.4; 5 mM MgCl 2 ; 1 mM PMSF and was incubated in ice for 20 min.Subsequently, SDS (2%) and β-mercaptoethanol (5%, v/v) was added.Following centrifugation at 13000 rpm for 20 min at 4°C, supernatant was transferred to new vial and was used in Bradford protein assay.Total soluble proteins (50 µg) were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and immunoblotted, using antibodies raised against the purified Arabidopsis P5CS protein.

Expression of Arabidopsis P5CS cDNA in transgenic tobacco plants
Following the reverse transcription and amplification, Arabidopsis P5CS was cloned in Pbluescript SK - andsubsequently subcloned in pBI121 by replacing Vigna P5CS (Figure 1).This clone along with two other P5CSs obtained from Vigna (pBI-P5CSs with and without feedback inhibition) was used for subsequent analysis.As it is well known, 35S promoter was originally derived from cauliflower mosaic virus, a Brassicaceae specific infecting virus.Thus, we hypothesized that 35S promoter may also promote Arabidopsis P5CS as efficient.Formerly, Kishor et al. (1995) have reported transformation of tobacco plant with the Vigna P5CS cDNA under 35S promoter control.In the present study, the same vector was used except that Vigna P5CS was substituted with Arabidopsis P5CS and then transformed into tobacco.

Molecular characterization of transgenic tobacco plants
In order to verify that the gene of interest was successfully cloned, expressed and translated, molecular analysis on the T 2 transgenic plants were carried out at three levels including DNA amplification, southern blotting along with RT-PCR and immunoblotting.Results (Figure 3) showed that, all samples including the wild type tobacco were reacted with the probe, indicating that Vigna P5CS cDNA not only hybridizes with Arabidopsis P5CS (pBI-P5CS X, either as an isolated vector or integrated into tobacco genome), but also with putative endogenous tobacco P5CS.According to this figure, three copies of P5CS cDNA were integrated with the genome of the plants.Based on Arabidopsis P5CS cDNA restriction map (Figure 1), Sac I and Bcl I restriction enzymes liberate a 1500 bp fragment from 5' terminus of the P5CS cDNA (Figure 3; lane 3).This fragment was detected in all transgenic tobacco plants.In addition, three bands with different sizes were also detected in T 2 transgenic plants (indicated with arrows), suggesting the insertion of three repeats of transfer DNA.The shorter bands in both transformed and non-transformed plants (Figure 3, lanes 1, 2) represent the endogenous genomic tobacco P5CS.
To verify whether exogenous P5CS is expressed and translated in transformed plants, immunoblotting was done using polyclonal antibody raised against Arabidopsis P5CS in rabbits.Figure 4  Table 1.Proline content of fresh leaves from control and P5CS transgenic T0 plants grown under normal and after five days of drought stress conditions (standard errors in parenthesis).sion of P5CS in non-transgenic plant in normal (nonstress) condition could not be detected, the expression of P5CS in transgenic plant under the same condition was relatively high and detectable as a 77-kDa band at the expected size in western blot analysis.

Assessment of T 0 transgenic plants proline content and generation of T 1 and T 2 transgenic plants
Results obtained in this part of study indicated that, proline content of transgenic plant in non-stress and stress conditions was much higher than control plants (Table 1).The higher proline content of transgenic plants under well watered condition could be attributed to the basal 35S promoter activity.On the other hand, following to five days of drought, no significant increase in proline content was detected in the control plant which is in contrast to the previous report.This difference in results might be explained to be due to the duration of applied stress.On the other hand, following to this period of stress, proline content in transgenic plants (except pBI P5CS X6) was increased two to three fold (Table 1), which is in agreement with the previous observations (Kishor et al., 2005a).To investigate expression status of the exogenous P5CS in T 0 plants, DNA and RNA were extracted from leaves of transgenic X9 plant and PCR or RT-PCR was carried out under the conditions explained earlier.
Interestingly, it was found that not all cells were transformed with exogenous P5CS (Figure 5).Leaves with negative PCR response were also negative for GUS assay (data not shown).Also, as Table 1 points out, among transgenic plants, proline content of leaves were significantly different (p<0.01).This inconsistency within transgenic T 0 plants may be caused by the heterogeneity in terms of P5CS expression.Since transgenic plants were generated by shooting from callus, it was possible that both transformed and non-transformed cells were present in the same shoot.Non-transformed cells could grow and reproduce probably because of degradation of kanamycin in the medium by neighboring transformed cells.Such phenomenon was also previously reported by other groups (Sherman et al., 1998).

Verifying P5CS expression pattern in transgenic plant
Under normal non-stress condition, the leaves of T 2 generation of pBI P5CS X 9 contained 1194 µg proline per gram fresh leaves, a value not significantly different (p-1 2 3 4 5 6 7 8 765 bp  value) from former published data (1000 µg per gram fresh leaf (Kishor et al., 1995) (Table 2).Low levels of proline content have also been observed in other plants in normal condition (Hmida-Sayari et al., 2005).They estimated 200 proline µg per gram of fresh leaves of P5CS transformed potato plant.Increasing salt concentration up to 200 mM in our study has resulted to enhanced proline content up to 2938 µg per gram fresh leaves which again was comparable to the results shown elsewhere (2600 µg per gram fresh leaf; (Table 2) for tobacco and 1126 µg per gram fresh leaves for potato in response to 180 mM salt (Hmida-Sayari et al., 2005).
Based on these results, it could be concluded that 35S promoter may promote expression of both types of P5CS genes (Vigna versus Arabidopsis) to the same extent regardless of the origin of the genes or their natural relationship to the promoter.It is well known that 35S is a strong promoter (Hammond et al., 2000), but whether the stated suggestion could be extended to weak or inducible promoters remains to be explored.

Expression of P5CS, germination and growth of plants under salt stress
Using PCR, we found that cells from T 2 plants were homozygous for exogenous P5CS and stably expressed the transformedArabidopsis P5CS cDNA.Following the evaluation of T 2 plants for salt tolerance, seeds from these plants were used for evaluating growth and germnation pattern under normal and stress conditions.Results obtained on days 1, 3, 7 and 16 after onset of germination was recorded in four different tables for analysis of variance.There was no significant difference between control and transgenic plants for germination on day one.However, the differences were significant (P < 0.0001) for days 3, 7 and 16 (Table 3a, b; Figure 6b), similar to the reports of Gelvin et al. (1989) on rice germination or Zhang et al. (2010) on barley germination and indicated a gradual increase in proline content of plants during the period of stress.Results showed that, control plants and plants transformed with vector (pBI121) were both similarly able to tolerate up to 100 mM NaCl in growth (Figures 7 and 8; Table 2a) and up to 50 mM NaCl in germination phases, respectively (Figure 6a, b; Table 3a).In contrast, transgenic plants were tolerant up to 250 mM NaCl in growth and 200 mM in germination phases, respectively (Figures 6 and 7; Tables 2a and 3a).Therefore, it was concluded that both transgenic and nontransgenic plants were more sensitive to salt stress during germination phase than to the growth phase.
A comparison of proline content of control and pBI121 transformed plants from 0 mM up to 100 mM of NaCl, revealed no significant differences (Figure 7; Table 2a).However, transgenic plants showed a significant difference in proline content not only for this range of salt    concentration but also from 100 to 250 mM of salt where control plants were no more tolerant (Figure 7). Figure 9a shows the response of control and transgenic plants to two weeks of drought stress and Figure 9b to 250 mM of salt for three weeks.As Figure 9a indicates, while control wild type plants display sensitivity to the drought as could be observed by the higher degree of wilting, transgenic plants are more resistant.On the other hand, up on application of salt stress we observed retardation in the growth and more deformation of leaves in the wild type plants.
Under normal condition, the proline content of transgenic plants was 7.5 times more than that of control plants (1194.04µg of proline per gram fresh weight of P5CS transformed plants versus 165.53 µg of proline per gram fresh weightof controlplants).This findingwas inaccordance with Kishor et al. (1995), who reported 1000 µg proline per gram fresh weight of transformed plants and 100 µg per gram fresh weight of control plants.How-ever, our results are not in agreement with the report by Yamada et al. (2005), who observed 1.3 to 3.5 folds of proline contents in Petunia with respect to the osmotic stress.On the other hand, transgenic plants responded to the salt stress by 2.5 to 3.3 folds further increase in proline content compared with the same plants under normal non-stress condition.
The amount of proline was measured to be 2938 up to 3836 µg per gram fresh weight of transformed plants in salt stress compared with the 1194 µg of proline per gram fresh weight in non-stress condition.
In addition to the Arabidopsis P5CS cDNA, two other Vigna P5CS vectors were used as positive controls.Among these two vectors, one displayed functional feedback inhibition by proline, whereas the other is not responsive as a result of disrupted feedback inhibition following to site directed mutagenesis (Kishor et al., 1995;Hong et al., 2000).Comparing salt tolerance for all three types of transformed plants (plants transformed with Arabidopsis P5CS cDNA and plants transformed with Vigna P5CS cDNAs; either with or without feedback inhibition) we observed the uppermost tolerance up to 250 mM of salt.From these observations, it was concluded that increase in proline level could confer plants withstanding up to specific concentration of salt.One cause for the observed similarity could be partly, due to 35S promoter activity that drives expression of both types of P5CSs.The other cause could be structural and functional similarities between two types of P5CSs that demands further future investigations.
In conclusion, our study indicates achievement of transformed plants that stably expressed P5CS up to three generations.Our further analysis on the fourth generation also supports this stability (data not shown).Comparing to the wild type plants, the transformed plants are tolerant up to four folds of salt concentrations.The successful maintenance of P5CS expression for several generations in model plants with successful transformation could be helpful in obtaining crops tolerant against osmotic stresses.

Figure 1 .
Figure 1.Vector used for expression of Arabidopsis P5CS cDNA in transgenic tobacco.The map of construct designed and used in Agrobacterium mediated tobacco plant transformation; pBI-P5CS X was constructed by replacing Vigna P5CS cDNA with double stranded cDNA of P5CS derived from A. thaliana.The entire cassette of the vector in both of constructs is the same.

Figure 2 .
Figure 2. (A) Genomic PCR on DNA extracted from a pBI-P5CS X9 transformed tobacco plant with primers designed such that upstream primer anneals inside 35S promoter and downstream primer within P5CS giving rise to 765 bp amplification product.Lane 1, Molecular size marker gene ruler 100 bp (Roche); lanes 2-5, PCR product of T2 transgenic plant (pBI-P5CS X9), lane 6, PCR product of a non-transgenic plant; (B) RT-PCR of RNA isolated from P5CS transformed plant line resulting to amplification of 634 bpcDNA.Lane 1, Molecular size marker gene ruler 50 bp (MBI, Fermentas); lane 2, negative control; all PCR reagent plus water instead of T2 transgenic cDNA (pBI-P5CS X9), lane 3, wild type plant cDNA as negative control; lane 4, RT-PCR of the plant transformed with pBI121 as negative control; lane 5, further negative control where RNA instead of cDNA was used for checking the possible DNA contamination with the extracted RNA; lane 6, RT-PCR of T2 transgenic cDNA (pBI-P5CS X9); lane 7, molecular size marker gene ruler 50 bp (MBI, Fermentas).
to a 765 bp PCR product from 5 ′ side of the P5CS.In addition, for limiting PCR amplification only to the cloned P5CS, the forward primers were designed to start few nucleotides before the end of one exon and the start of the next exon.Accordingly, amplification was restricted only to the reverse transcribed cDNA of the cloned P5CS.The sequence of the second set of primers was the forward; 5
indicates that, the expression of P5CS is limited to the transformed plants.While expres-

Figure 5 .
Figure5.Genomic PCR on the DNA extracted from a pBI-P5CS X9 transformed tobacco plant (T0 plant) with primers designed such that the upstream primer anneals within 35S promoter and the downstream primer within internal P5CS sequence, giving rise to 765 bp amplification product.lane 1, Negative control; All PCR reagent plus water instead of DNA; lane 2, PCR product of a nontransgenic plant; lane 3, PCR product of plant transformed with pBI121; lane 4, PCR product of T0 transgenic plant (pBI-P5CS X9); lane 5, molecular size marker gene ruler 100 bp (MBI, Fermentas); lanes 6 and 7, repeat of PCR on the DNA extracted from T0 transgenic plant (pBI-P5CS X9).A negative result in lane 6 could be interpreted as a result of mosaic phenomenon in the transformation; lane 8, PCR product on the plasmid contain Arabidopsis P5CS cDNA as positive control.

AFigure 6
Figure 6.(A and B) Germination profile and percent of T2 generation transgenic versus wild type seeds under normal and salt-stress condition.

Figure 7 .
Figure 7.Histogram of proline assay on T2 generation transgenic and wild type plants under normal and salt stress conditions.Means with non-overlapping sets of alphabets differ significantly from each other.No growth could be observed for wild type and pBI 121 transformed plants in 200 and 250 mM of salt concentration.

Figure 9 .
Figure 9.Growth pattern of T3 transgenic versus control plants exposed to 15 days of drought (A); or 21 days of exposure to 250 mM salt (B).

Table 2a .
Percent of germination of T2 generation transgenic and wild type plants under normal and salt-stress conditions (standard errors in parenthesis).

Table 2b .
Analysis of variance for percent germination of T2 generation transgenic and wild type plants on day sixteen under normal and salt-stress condition.
DF, Degree of freedom.

Table 3a .
Least-square estimates of proline contents (µg/g in vitro fresh leaves) in T2 generation transgenic plants, tobacco transformed with pBI121 and wild type plants under normal and salt-stress conditions (standard errors in parenthesis).

Table 3b .
Analysis of variance for proline contents (µg/g in vitro fresh leaves) in T2 generation of transgenic plants, tobacco transformed with pBI121 and wild type plants under normal and salt-stress conditions.