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South African Journal of Botany 73 (2007) 505 – 511

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Transformed tobacco plants with increased tolerance to drought

J. Gubiš a,⁎, R. Vaňková b , V. Červená a , M. Dragúňová c , M. Hudcovicová a , H. Lichtnerová c ,
T. Dokupil c , Z. Jureková d
a
Slovak Agricultural Research Centre, Research Institute of Plant Production, Bratislavská cesta 122, 921 68 Piešt'any, Slovak Republic
b
Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany ASCR v.v.i., Rozvojová 263, 165 02 Praha 6, Czech Republic
c
Laboratory of Explantate Cultures, Horticulture and Landscape Engineering Faculty, Slovak University of Agriculture, Tulipánová 7, 946 76 Nitra, Slovak Republic
d
Department of Ecology, Faculty of European Studies and Regional Development, Slovak University of Agriculture, Mariánska 10, 949 76 Nitra, Slovak Republic

Received 8 November 2006; received in revised form 29 March 2007; accepted 31 March 2007

Abstract

P5CSF129A cDNA and the nptII marker gene were used for tobacco (Nicotiana tabacum L. cv. Bel B and cv. M51) transformation via
Agrobacterium tumefaciens strain LBA4404. Twenty transformed tobacco plants were obtained after transformation of leaf discs. Presence of
the transgene was confirmed by polymerase chain reaction (PCR) analysis. Physiological responses to water stress were compared in transgenic
and wild-type tobacco plants. Transgenic plants of both cultivars accumulated high levels of free proline. They did not exhibit dry mass relocation
or chlorophyll content reduction. Neither precocious senescence, nor leaf necrosis or morphological changes were observed in control and stress
conditions (RWC decrease by 7–8%). Transgenic plants with elevated accumulation of osmoprotectants seem to be better adapted to water stress,
providing a perspective for future research of stress effects that have a principle role in the functional activity of plants. This study confirmed
P5CSF129A to be a candidate gene in crop engineering for enhanced water stress tolerance.
© 2007 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Nicotiana tabacum L.; Pigment; Proline; Relative water content; Stress

1. Introduction The effectiveness of osmotic adjustment by osmoprotectants

was proved using transgenic plants (Bohnert and Jensen, 1996;
Plants are often exposed to various adverse environmental Nuccio et al., 1999; Rontein et al., 2002), which accumulated
stresses such as drought, salinity, high and low temperatures. higher concentrations of proline, glycinebetaine, or pinitol
The organisms respond to the change of environmental (Kishor et al., 1995; Zhang et al., 1995; Sheveleva et al., 1997;
conditions by activation of enzyme reaction products which Takabe et al., 1998). An alternative strategy is regulation of
contribute to the re-establishment of homeostasis. During water water permeability through water channels, which may also
stress, lower and higher plants accumulate osmoprotectants, modify the sensitivity to water stress (Aharon et al., 2003).
such as glycinebetaine and proline (Gorham et al., 1985; Overexpression of the Δ1-pyrroline-5carboxylate synthetase
Delauney and Verma, 1993). The typical levels of naturally gene (P5CS) encoding the first enzyme of the proline
accumulated osmoprotectants are 5–50 μmol/g of fresh weight biosynthesis pathway was reported to enhance proline produc-
(∼ 6–60 mM on a plant water basis), maxima are reached during tion in transgenic plants (Kishor et al., 1995). The level of
exposure to osmotic stress (Rhodes and Hanson, 1993; Bohnert proline can be increased 2-fold (Hong et al., 2000) provided that
et al., 1995). the feedback inhibition by proline is removed. This was done by
Zhang et al. (1995), who used the site directed mutagenesis in
order to replace Phe at position 129 by Ala in Vigna aconitifolia
P5CS gene (P5CSF129A).
⁎ Corresponding author. Recently, successful attempts have been made to transfer
E-mail address: gubis@vurv.sk (J. Gubiš). genes involved in stress tolerance into plants. The resulting
0254-6299/$ - see front matter © 2007 SAAB. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.sajb.2007.03.011
506 J. Gubiš et al. / South African Journal of Botany 73 (2007) 505–511

transgenic plants were reported to exhibit enhanced tolerance to Dellaporta et al. (1983). PCR analysis was performed using specific
water stress and other stresses (Xu et al., 1996; Hsieh et al., primers to detect P5CSF129A gene (forward 5′-GTGAAGA-
2002; Bhattacharya et al., 2004; Chandra Babu et al., 2004). TAACGCCTGGAGC-3′and reverse 5′- GTTTTCCACCAACAT-
However, information about physiological reactions of mod- TATCTG-3′) amplifying an amplicon of 468 bp and nptII gene
ified plants is still very limited. (forward 5′-CAGACAATCGGCTGCTCTGAT-3′and reverse 5′-
Tobacco is an important model plant that has been TGCGAT GTTTCGCTTGGTGGT-3′) amplifying an amplicon of
extensively used for the study of genetic, physiological and 330 bp. The PCR procedure was performed as follows. DNA (1 ml)
metabolic processes (Elleuch et al., 2001), ranging from tissue was added to a final volume 15 ml with 0.3 ml of each forward and
culture establishment, via floral growth and development reverse primers in concentration 0.2 μM, 1× PCR buffer (20 mM
(Mandel et al., 1992) to the evaluation of the impact of Tris–HCl pH 8.4, 50 mM KCl, 1.5 nM MgCl2), 0.2 mM of dNTP,
environmental pollution (Monciardini et al., 1998). The aim of 0.8 units of Taq DNA polymerase, and 25 ng of DNA. PCR with the
our study was transformation of tobacco leaf discs with a gene P5CSF129A gene was performed in GeneAmp® PCR System 9700
improving drought tolerance and evaluation of the stress (Applied Biosystems) under the following conditions: at 94 °C for
response and adaptation ability of the resulting transformed 4 min, 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min,
tobacco. and extra extension at 72 °C for 8 min. PCR with the nptII gene was
performed at 94 °C for 5 min, 30 cycles at 94 °C for 1 min, 55 °C for
2. Materials and methods 1 min, 72 °C for 45 s, and extra extension at 72 °C for 8 min.
Electrophoretic detection of PCR products was performed in 1.4%
2.1. Plant material: in vitro explants agarose gels stained with ethidium bromide.

Seeds of Nicotiana tabacum L. cultivars Bel B and M51, 2.4. Histochemical analysis
respectively, were surface-decontaminated by shaking in 70%
ethanol for 1 min and in 4% sodium hypochlorite for 10 min. The expression of GUS gene was detected by histochemical
Subsequently they were rinsed four times with sterile distilled water. staining according to Jefferson et al. (1987). Leaf segments of
The seeds were then allowed to germinate in glass containers with transgenic plants were immersed in 1 mM 5-bromo-4-chloro-3-
25 cm3 of MS-based medium (Murashige and Skoog, 1962). The indolyl-β-D-glucuronic acid (X-GlcA) solution at 37 °C over-
cultures were initially kept in the dark at 27 ± 1 °C for 7 days and night and rinsed with 70% ethanol. The expression of the GUS
then maintained under a photoperiod of 16 h illumination with a gene was inspected microscopically. The transient expression
light intensity of 50 μmol m− 2 s− 1, with day/night temperatures of efficiency of the GUS gene was calculated as the number of
25 °C/20 °C. positive segments compared to the number of segments tested.

2.2. Transformation via Agrobacterium tumefaciens 2.5. Stress treatment

A plasmid (pBI-P5CSF129A) containing mutagenized V. Rooted plantlets were acclimatised for 4 weeks and then
aconitifolia P5CSF129A cDNA (Zhang et al., 1995) under the transplanted to pots with soil. The plants were grown in
control of the cauliflower mosaic virus 35S promoter, and greenhouse at defined temperature (25–30 °C) and relative
neomycine phosphotransferase (nptII) marker gene was used for humidity (65–85%) in summer. Plants at the stage of 5 leaves
tobacco transformation via Agrobacterium tumefaciens strain (wild type cv. M51 and cv. Bel B as well as the transformed
LBA4404. For tobacco transformation, 8-week-old leaves were ones) were divided into 2 groups: control (70% water content in
cut into 50 × 50 mm discs, inoculated with bacterial suspension soil) and plants stressed by drought (40% water content in soil).
at OD600 = 0.6 for 15 min and blotted dry with sterile filter paper After the 40% water content in soil was reached, this level of
to remove excess bacterial suspension. The inoculated leaf discs water stress was constantly maintained. The first analysis of
were transferred to MS-based medium containing 1 mg dm− 3 samples was made before inducing stress, and then on days 12
BAP, and kept in the dark for 3 days. After co-cultivation, the and 18 after treatment. The following parameters were
transformed leaf discs were transferred to shoot regeneration quantified: fresh (FW) and dry (DW) weights of plants, relative
medium supplemented with 1 mg dm− 3 BAP, 100 mg dm− 3 water content, content of free proline and content of pigments.
kanamycin, 250 mg dm− 3 cefotaxime and 250 mg dm− 3
carbenicillin under the photoperiod as described above. Eight 2.6. Proline determination
weeks later, when regenerated plantlets reached 20–30 mm in
height, they were cut off and placed on a selective rooting The proline content was determined in leaf samples (1 g
medium (MS-based medium + 100 mg dm− 3 kanamycin). FW) according to Bates et al. (1973) by measuring the
Rooted plantlets were transferred to ex vitro conditions. quantity of the coloured product of proline reaction with
ninhydric acid. The absorbance was read at 519 nm using a
2.3. DNA extraction and PCR analysis Spectroquant® Spectrophotometer NOVA 400 (Merck Ltd.).
The proline concentration was determined from a standard
For molecular analysis of transgenic tobacco, plant genomic curve and calculated on a fresh weight basis (μmol proline
DNA was extracted from 1.5 g fresh plant tissue according to g− 1 FW).
 J. Gubiš et al. / South African Journal of Botany 73 (2007) 505–511 507

2.7. Determination of relative water content (RWC) acclimatisation. No regeneration of control (untransformed)
explants was observed on regeneration medium supplemented
Leaves collected for determination of the relative water with 100 mg dm− 3 kanamycin (data not shown).
content (RWC = [(FW − DW) / (FWsaturated − DW)] × 100%) were After selection and multiplication under in vitro conditions,
of similar physiological age as those collected for proline the regenerated shoots were tested for the presence of
determination. The leaves were re-cut in water, and kept in introduced genes using PCR analysis. Also the rooted shoots
distilled water in a closed glass flask at room temperature transplanted to soil (Fig. 1b, c) were subjected to PCR analysis
(21 °C). The FW of the leaves was determined before and after which confirmed the presence of both — nptII and P5CSF129A
incubating for 5 h at room temperature. Leaves were then dried genes. The presence of both genes was confirmed in all tested
for 48 h at 65 °C to determine DW. samples originating from in vitro cultures as well as in ex vitro
acclimatised plants before and after the stress treatment (Fig. 2).
2.8. Determination of pigment contents The in vitro rooted transformants were also tested using the
histochemical GUS assay. All the regenerated shoots proved to
The content of chlorophyll a, b and carotenoids in leaves be GUS-positive (Fig. 3).
was determined after extraction in 80% acetone at 20 °C. The synthesis and accumulation of free proline was already
Absorption was measured at 663 nm, 645 nm and 440 nm using established in the in vitro conditions by means of quantifica-
a Spectroquant® Spectrophotometer NOVA 400 (Merck Ltd.). tion of free proline content in the organ culture. Explants of
The leaf samples were of similar physiological age as those transgenic cv. M51 accumulated free proline in quantities of
collected for proline and RWC determination. 8.05 μmol/g FW, in comparison with 0.37 μmol/g FW
detected in wild-type M51. The transgenic cv. Bel B
2.9. Statistics accumulated 12.75 μmol/g FW (0.34 μmol/g FW was
detected in wild-type Bel B). A similar pattern of free proline
The data presented in this paper is the mean from two accumulation was found in plants grown in a greenhouse
independent experiments. All data were analysed for signifi- (transformed ones in comparison with the wild-type), under
cance (P ≤ 0.05) by ANOVA with mean separation by Duncan's both conditions — sufficiently supplied with water and
test using statistical software SPSS (13.0). subjected to water deficit. Significant differences (P ≤ 0.05) in
proline content were detected also among the genotypes used.
3. Results and discussion On average, free proline accumulation in leaves of trans-
formed tobacco plants in control conditions (70% water
The aim of our study was to obtain regenerated tobacco content in the soil) was almost 17-fold (cv. M51) and 22-fold
shoots transformed with proline biosynthetic gene in order to (cv. Bel B) higher than in the wild-type lines. The free proline
improve plant tolerance to stresses. A total of 40 explants of accumulation in leaves under stress conditions (40% water
tobacco cv. Bel B and cv. M51, respectively, were transformed content in the soil) was found to be 14-fold (cv. M51) and 16-
with A. tumefaciens carrying plasmid with gene P5CSF129A. fold (cv. Bel B) higher when compared to stressed wild-type
Five putatively transformed shoots of cv. Bel B and 15 of cv. plants (Fig. 4). Under stress conditions, proline accumulates
M51 were cultivated on selective medium (Fig. 1a). Regenera- in higher quantities in leaves of cv. M51 than in the leaves of
tion frequency of cv. Bel B and cv. M51 was 50% (1.33 shoots cv. Bel B.
per regenerated explant) and 52.5% (1.4 shoots per regenerated Overproduction of proline in plants may increase the
explant), respectively. All plants transplanted to soil survived tolerance to abiotic stresses. Kishor et al. (1995) showed that

Fig. 1. Transgenic tobacco transformed with A. tumefaciens LBA4404 carrying the nptII and P5CSF129A genes from V. aconitifolia. (a) Shoot regeneration on leaf-
discs of N. tabacum L. cv. M51, (b) regenerated shoot after rooting on selective medium, (c) L0 line of transgenic tobacco.
508 J. Gubiš et al. / South African Journal of Botany 73 (2007) 505–511

Fig. 2. Molecular analysis of L0 lines of transgenic tobacco. (A) PCR analysis using P5CSF129A gene primers, M: 50 bp ladder marker (Invitrogen Life Technologies,
USA); lane 1: water as negative control; lane 2: DNA of pBI-P5CSF129A plasmid as positive control; lanes 3–6: transformed tobacco lines from ex vitro plants; lanes
7–10: transformed tobacco from in vitro conditions. (B) PCR analysis using nptII gene primers, M: 50 bp ladder marker (Invitrogen Life Technologies, USA); lane 1:
water as negative control; lane 2: DNA of pBI-P5CSF129A plasmid as positive control; lanes 3–6: transformed tobacco lines from ex vitro plants; lanes 7–10:
transformed tobacco from in vitro.

proline accumulation correlated with tolerance to drought and Except the P5CS gene, other genes were exploited in
salinity stresses in plants. Transgenic plants of tobacco prepared tobacco transformation to improve plant tolerance to drought
by Kishor et al. (1995) synthesized 10- to 18-fold more proline stress e.g. TPS1 gene (Romero et al., 1997), otsA and otsB
than control plants. The osmotic potential of leaf sap from genes (Goddijn et al., 1997), lp3 gene (Wang et al., 2003), Asr1
transgenic plants was less decreased under water stress gene (Kalifa et al., 2004), BoRS1 gene (Tang et al., 2005),
conditions compared to those of control plants. Overproduction AtTPS1 gene (Almeida et al., 2005), CAP2 gene (Shukla et
of proline also enhanced root biomass and flower development al., 2006) and NtHSP70-1 drought-/ABA-inducible gene (Cho
in transgenic plants under drought stress conditions. Accumula- and Hong, 2006).
tion of proline in plants under stress may offer multiple benefits Physiological characterization of the studied genotypes
to the cell. A role of proline in scavenging free radicals in cells revealed different abilities of biomass production. Cultivar
exposed to salinity has been clearly shown by Hong et al. Bel B had a slightly higher capability of dry mass
(2000). These authors also studied the proline biosynthesis production; however, it was more sensitive in stress
regulation. They showed that removal of feedback inhibition in conditions. Transformation did not have negative effects on
P5CSF129A resulted in a 2-fold increase in proline content the dynamics of dry mass production especially in cv. Bel B
compared with that in the plants expressing V. aconitifolia wild- (Fig. 5). Under stress conditions, dry mass was reduced less
type P5CS under both normal and stress conditions. This in transgenics than in wild type plants. The improvement of
difference was further increased in plants treated with 200 mM water stress tolerance, which we observed in our transgenic
NaCl. tobacco plants with the elevated osmoprotectant content, is

Fig. 3. Histochemical GUS assay of a transformant. Blue precipitations represent the activity of the GUS gene.
 J. Gubiš et al. / South African Journal of Botany 73 (2007) 505–511 509

of water supply resulted in a slow decline of RWC. Two

percent decline in the leaf RWC was determined in wild-type
M51 as well as in transgenic Bel B after 12 days of water
stress, whilst in transgenic M51 and wild-type Bel B the leaf
RWC declined by 5%. After 6 more days of water stress, a
decline by approximately 4–5% in leaf RWC in wild-type M51
and Bel B and 7–8% in transgenic plants Bel B and M51 was
observed.
Exposure of the tobacco plants to water stress for 12 and 18
days resulted in the elevation of the content of Chl a, Chl b, total
Chl and carotenoids (Table 1). Statistically significant differ-
ences (P ≤ 0.05) in the total Chl and carotenoids were found
Fig. 4. Proline content in wild-type and transgenic tobacco leaves in control (soil between cultivars as well as during deficit progression. The
water content 70% — black bars) and stress (soil water content 40% — white content of total Chl and carotenoids was particularly affected by
bars) conditions. Data represent the mean values from three offtakes (n = 6).
genotype. In wild-type cv. Bel B the total Chl and carotenoid
Duncan's test at 5% level of significance. The error bars show SE.
contents increased by about 31 and 36%, respectively, after
12 days of water stress treatment. Increase in the total Chl and
in accordance with the results of Delauney and Verma carotenoid contents continued to 44 and 34%, respectively,
(1993). recorded after 18 days of water stress treatment. In transgenic cv.
Increase of abiotic stress tolerance in model plants and Bel B the total Chl and carotenoid contents increased by about
presumably in other crops over-expressing the AtTPS1 gene 52 and 63%, respectively after 12 days of water stress treatment,
(involved in trehalose biosynthesis in Arabidopsis thaliana) whilst a 47 and 37% increase, respectively in the total Chl and
was suggested by Almeida et al. (2005), who observed higher carotenoid contents was recorded after 18 days of water stress
germination rates under both osmotic and temperature stress in treatment. In wild-type M51 total Chl and carotenoid contents
tobacco plants containing AtTPS1 gene. increased by about 2 and 10%, respectively, after 12 days of
In this study, leaf relative water content (RWC) was also water stress treatment. Eighteen days after the water stress
followed. The control plants had 86–94% of RWC. Cessation treatment the content of both total Chl and carotenoids increased

Fig. 5. The effect of water stress on dry mass accumulation in leaves of wild-type and transgenic tobacco plants at 0, 12 and 18 days of water stress treatment. The bars
show standard errors.
510 J. Gubiš et al. / South African Journal of Botany 73 (2007) 505–511

Table 1
Photosynthetic pigment content [mg m− 2 (FW)] in tobacco cvs., 0, 12 and 18 days after water stress treatment
Treatment 0 day 12 days 18 days
Cultivar Water content Chl a Chl b Total Chl Carotenoids Chl a Chl b Total Chl Carotenoids Chl a Chl b Total Chl Carotenoids
in soil (%)
w.t. M51 40 198.38 73.13 271.45a 62.58a 198.68 77.94 276.55bc ⁎ 68.82b ⁎ 215.72 170.73 316.37cd ⁎ 70.82d
70 165.55 63.32 228.81a 54.39a 192.87 80.98 273.78b 60.89bc
M51 40 200.49 74.14 274.57a 62.00a 200.16 73.82 273.91bc 63.80ab 186.80 72.48 259.22ab 58.3b ⁎
70 174.60 60.21 234.75ab 56.95a 170.00 67.72 237.65a 51.51a
w.t. Bel B 40 166.24 61.72 227.90a 50.83a 214.47 83.98 298.37c 69.11b 230.89 98.27 329.08cd 68.03d
70 207.81 81.51 289.25c 68.36b 215.08 92.47 307.47c 65.4cd
Bel B 40 169.72 59.55 229.21a 50.58a 250.55 97.74 348.20d 82.22c ⁎ 235.73 101.41 337.05d 69.49d ⁎
70 218.02 93.85 311.79cd 66.42b 194.41 85.78 280.12b 57.37b
⁎ Significantly different from control (70% water content) at P ≤ 0.05. Superscript letters mean significant difference at P ≤ 0.05.

by about 17 and 13%, respectively. Significant difference was Agency VEGA of the Ministry of Education, Slovakia, and by
also found in transgenic cv. M51, with the increase of carotenoid the Slovak Academy of Sciences.
content by about 3% after 12 days of water stress treatment
(Table 1). The effect of drought stress on chlorophyll content References
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Edited by E Balazs