Evaluation of salt tolerance in ectoine-transgenic tomato plants ...

RESEARCH PAPER

GM Crops 2-1, 58-65; January/February/March 2011; © 2011 Landes Bioscience

Evaluation of salt tolerance in ectoine-transgenic tomato plants (Lycopersicon esculentum) in terms of photosynthesis, osmotic adjustment and carbon partitioning Reda E.A. Moghaieb,1,* Akiko Nakamura,2 Hirofumi Saneoka2 and Kounosuke Fujita2 Department of Genetics; Faculty of Agriculture; Cairo University; Giza, Egypt; 2Department of Environmental Dynamics and Management; Graduate School of Biosphere Sciences; Hiroshima University; Higashi-Hiroshima, Japan

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Key words: ectoine, osmotic potential, oxidative stress, photosynthesis, transformation, tomato plants

Ectoine is a common compatible solute in halophilic bacteria. Its biosynthesis originates from L-aspartate β-semialdehyde and requires three enzymes: L-2,4-diaminobutyric acid aminotransferase (gene: ect B), L-2,4-diaminobutyric acid acetyl transferase (gene: ect A) and L-ectoine synthase (gene: ect C). Genetically engineered tomato plants expressing the three H. elongata genes (ectA, ectB and ectC) generated showed no phenotypic abnormality. Expression of the ectoine biosynthetic genes was detected in the T3 transgenic plants by northern blot analysis. The ectoine accumulating T3 plants were evaluated for salt tolerance by examining their photosynthestic activity, osmotic adjustment and carbon partitioning. Nuclear magnetic resonance (NMR) detected the accumulation of ectoine. The concentration of ectoine increased with increasing salinity. The transgenic lines showed higher activities of peroxidase, while the malondialdehyde (MDA) concentration was decreased under salinity stress condition. In addition, preservation of higher rates of photosynthesis and turgor values as compared to control was evident. Within a week of 13CO2 feeding, salt application 13 led to increases in the partitioning of 13C into roots atL the expense ofi Cc in the other plant parts. These results suggest ©2 01 1 a nd esBi os c en e. that under saline conditions ectoine synthesis is promoted in the roots of transgenic plants, leading to an acceleration Donotdi s t r i but e. of sink activity for photosynthate in the roots. Subsequently, root function such as water uptake is improved, compared with wild-type plants. In this way, the photosynthetic rate is increased through enhancement of cell membrane stability in oxidative conditions under salt stress.

Introduction Soil salinity is one of the major environmental factors contributing to loss of agricultural land and reduced crop productivity worldwide.1 The detrimental effects of salt on plants are a consequence of both water deficits resulting in osmotic stress and the effects of excess sodium ions on key biochemical processes. In response to salinity stress, plants accumulate low-molecular weight osmolytes such as sugar alcohols (e.g., glycerol, sorbitol and mannitol), and specific amino acids (proline and the quaternary ammonium compound glycine betaine). The cultivated tomato (Lycopersicon esculentum) is moderately sensitive to salinity, although some cultivars, such as ‘Money maker’ and ‘Edkawi’, perform well under moderately saline treatments.2 Salt-tolerant tomatoes can be developed by production of transgenic plants where osmoprotectants can be introduced into their genome. An important feature of osmoprotectants is that their beneficial effects are generally not species-specific and alien osmoprotectants can be introduced into plants to protect their

new host.3 Transgenic plants harboring genes for the biosynthesis of mannitol,4 proline,5 ononitol,6 trehalose,7 betaine,8-10 fructan11 and ectoine12,13 have proven more tolerant to salt stress than their wild-type counterparts. Salt stress adversely affects plant growth mainly by impairing photosynthetic activity as well as photoassimilate partitioning. Suwa et al.14 previously found that 13C partitioning among plant parts is inhibited earlier by salt application than photosynthetic rate in non-transgenic tobacco. Additionally, when photosynthate translocation is inhibited by salt, sugars deposited in the leaves may suppress the expression of genes encoding carboxylation enzymes, such as Rubisco,15 leading to depression of photosynthetic activity. However, the improved salt tolerance of ectoine-transformed plants has not yet been examined in terms of photosynthesis and photosynthate partitioning. Moreover, our previous data indicated that the uptake and translocation of nitrate-N in roots is impaired by salinity and that the impairment can be alleviated by accumulation of ectoine in the roots of ectoine-transformed tobacco plant.13 This result suggests that

*Correspondence to: Reda E.A. Moghaieb; Email: [email protected] Submitted: 02/26/11; Revised: 04/07/11; Accepted: 04/14/11 DOI: 10.4161/gmcr.2.1.15831 58

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Figure 1. Schematic representation of the pBI-101 Hm ect. ABC plasmid (A), Detection of ect C gene in the T3 plant genomes by PCR analysis: lane C is control and lanes 1–6 are ectoine transgenic plants (B). The analysis of ectoine gene expression by northern blots analysis (C). MWT is a control (nontransgenic Momotaro) and MT: ectoine transgenic plant of Momotaro and SWT: control (GUS transformed) and S1 and S2 are transgenic lines of Saturn, respectively.

and stem base at a much earlier time than photosynthetic activity of source leaf.16 Based on these findings, it is hypothesized that ectoine may improve nitrogen metabolism in roots by alleinhibition ©201 1L andesviating Bi os c i e nc e. of root sink activity as a response to salinity. Therefore, the present study is undertaken to test this hypothesis. Donotdi s t r i but e. Another important question to answer is which is more intensively and quickly impaired in the roots, sink activity for photosynthate or nitrogen metabolism? However, an answer to this question would still be inadequate for understanding why salt tolerance is improved by ectoine gene expression. Therefore, the present study was undertaken to transform tomato plants with ectoine biosynthetic genes and to evaluate the salt tolerance of the transformed plants in terms of biomass production and ectoine biosynthesis, photosynthesis, water status and 13C partitioning. Results and Discussion

Figure 2. Ectoine concentration in leaves and roots of transgenic (MT, S1 and S2) and control tomato plants.

root function, particularly concerning N metabolism, is affected. On the other hand, a tracer experiment of continuous chasing of 11C transport by PETIS (Positron Emitting Tracer Imaging System) has revealed that salinity impairs sink activity of roots

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In the present study, ectoine transgenic tomato plants were constructed. The hypocotyl explants isolated from two tomato cultivars were transformed with the A. tumefaciens LBA-4404 harboring the three genes involved in ectoine biosynthesis. Ectoine accumulating tomatoes (T3) were subjected to PCR analysis using Ect C gene specific primers. The PCR analysis indicated that clear band corresponding to the relevant sequence of the Ect C gene (432 bp) was detected in transgenic and absent in the control plants (Fig. 1B). Ectoine A, B and C gene expression can be detected only in the T3 plants (Fig. 1C). The ectoine accumulating tomatoes as well as the control plants were subjected to gradual increases in NaCl concentration (0, 100, 200 and 300 mM) for one week. Under salt stress condition, transgenic plants accumulate ectoine which is continuously increased with increasing NaCl concentration (Fig. 2). There are two possibilities for these increment in ectoine concentration,

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Figure 3. Effect of salinity on plant biomass in ectoine transgenic (lines MT, S1 and S2) and control (MWT and SWT) tomato plants. Values are means ± SE of 10 replicates.

Table 1. Effect of salinity on water relations in leaves of ectoine transgenic (T2 of lines MT, S1 and S2) and wild-type (MWT and SWT) tomato plants

©201 1L a ndesBi os c i enc e. OA Ψp (MPa) Donotdi s t r i but e.

Genotypes

NaCl mM

Ψw (MPa)

Ψs (MPa)

M WT

0

-0.962

-1.873

0.911

MT

S WT

S-1

S2

100

-1.005

-2.011

1.006

0.138

200

-1.054

-2.217

1.163

0.344

300

-1.324

-2.607

1.283

0.734

0

-0.785

-1.107

0.322

100

-1.275

-1.691

0.416

0.584

200

-1.397

-2.011

0.614

0.904

300

-1.471

-2.062

0.591

0.955

0.253

0

-0.637

-0.890

100

-0.809

-1.462

0.653

0.572

200

-1.150

-1.837

0.687

0.947

300

-1.079

-2.005

0.926

1.115

0

-0.711

-1.055

0.344

100

-0.809

-1.710

0.901

200

-0.956

-2.056

1.099

0.655 1.1

300

-0.539

-2.334

1.795

1.279

0

-0.588

-0.859

0.271

100

-0.785

-1.429

0.644

0.57

200

-0.932

-1.954

1.022

1.095

300

-0.686

-2.399

1.713

1.54

Seedling were planted into soil pots for 30 d, and then exposed to different NaCl treatments. On the sixth day of exposure, water potential (Ψw) and osmotic potential (Ψs) of the fifth expanded leaf counted from the uppermost leaf were measured. Turgor potential (Ψp) and osmotic adjustment (OA) were calculated as described in Materials and Methods. Values are means of five replicates.

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the first one is that, under salt stress condition the ectoine biosynthetic genes is post-transcriptionally regulated, another possibility could be that the degradation of ectoine is impaired by salt stress. These results agreed with previous findings regarding ectoine accumulation in transgenic tobacco plants.13 Under salinity condition, transgenic tomato plants accumulate much higher ectoine than the ectoine transgenic tobacco cell line previously reported by Nakayama et al. The data indicated that, increasing NaCl concentration reduced the plant dry weight, which was more severe in the control compared with the ectoine accumulating plants (Fig. 3). The transgenic MT line from the cultivar Momotaro showed a higher dry weight values followed by the transgenic S1 line that resulted from the cultivar Saturn at the same salt concentration (Fig. 3). The effect of salt stress on the photosynthetic activity was compared in ectoine expressing and control plants. The data indicated that saline conditions depressed the photosynthetic rate (Fig. 4). The MT transgenic line showed higher photosynthetic rate compared with the MWT (control), and similar trends were observed with S1 and S2 lines in relation to their corresponding wild type (SWT). This implies that salinity impairs plant biomass

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Figure 4. Effect of salinity on apparent photosynthetic rate in leaves of control (MWT and SWT) and ectoine transgenic, MT and S1 and S2 of Momotaro and Saturn, respectively. Seedlings were planted into soil pots for 30 d, and then exposed to the different NaCl treatments. The photosynthetic rates were measured daily in the fifth leaf from the uppermost leaf. Values are means of 10 replicates.

production by reducing its photosynthetic rate. Moghaieb et al.13 found that under salt stress ectoine expressing tobacco had higher photosynthetic rate compared with the control plants. The same observation was made regarding the amount of Rubisco, which was more suppressed in the control compared with ectoine expressing plants at 200 mM NaCl.13 The data showed that within a week of 13CO2 feeding, salt application led to increases in the partitioning of 13C into roots, at the expense of other plant parts (Fig. 5). However, a similar trend was not observed in the export rate (Fig. 6). The present data indicated that, under salt stress condition, ectoine synthesis is promoted in the roots of transgenic plants. This may lead to


the acceleration of sink activity in roots, thus improving water uptake in transgenic plants, compared with wild-type plants. In the present study, under salt stress ectoine accumulating plants could maintain higher ψp values compared with the control plants (Table 1). The higher ψp values of these transgenic plants may be the reason of why the transgenic plant growth is improved under salinity stress. An increase in peroxidase (POD) levels was observed in all salt-treated plants and the S2 transgenic line showed a higher POD activity at 200 mM NaCl compared with the S1 line and the MT line (Fig. 7). Similar results were obtained in studies in which hydrogen peroxide was found to accumulate under

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Figure 6. 13C export rate in control and ectoine transgenic tomato cv. Momotaro plants under salinity stress. Seedlings were planted into soil pots for 30 d, and then exposed to the different NaCl treatments. Six days after salinity treatment, tomato plant were fed for 13C. Twenty four hours after feeding, plants were taken and dissected into various plant parts. Values are means of three replicates. Figure 5. Effect of salinity on 13C partitioning to different plant parts of 30 ml of LB medium containing 50 μg/ml kanamycin sulfate, the control (wild-type) and ectoine transgenic tomato cv. Momotaro. then collected Values are means of three replicates. ©201 1L andesand Bi os c i en c e. by centrifugation at 1,120x g for 5 min. The

pellet was re-suspended in MS medium. The hypocotyl explants Donotdi s t r i but e.

hypoxic conditions in the roots and leaves of Hordeum vulgare17 and the leaves of barley.18 Malondialdehyde (MDA) is well documented to be accumulated in plant cell under salt stress as a result of lipid peroxidation through oxidative stress.19 So measurement of MDA concentration under salt stress can give an indication about the level of membrane damage.20-22 In the present study transgenic plants accumulated less amount of MDA in their leaves under salinity stress compared with the control plants, indicating that ectoine accumulation reduces the lipid peroxidition in transgenic compared with the control plants (Fig. 8). Taken together, these results indicate that ectoine synthesis was stimulated in the leaves and roots by salt application, which improved water status by maintaining higher activities of water uptake and transport to leaves. Furthermore, our results support the hypothesis that ectoine alleviates inhibition of root sink activity as a first response to salinity, as shown by the increase in 13C partitioning to roots. Materials and Methods Plant material and transgenic plants. Tomato seeds cv. ‘Momotaro’ and cv. ‘Saturn’ were germinated on Murashige and Skoog medium23 (MS). Hypocotyl explants were excised from 6-day-old seedlings and used for ectoine transformation. Agrobacterium tumefaciens strain LBA4404 cells harboring the binary Ti vector pBI101 Hm ect. ABC12 were grown overnight in

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prepared from 6-day-old seedlings were immersed in the bacterial suspension for 5 min. The explants were then blotted with sterilized filter paper and placed on a co-cultivation medium consisting of MS medium supplemented with 2 mg/l trans-zeatin. After co-cultivation for 3 d, the explants were transferred to a selection medium consisting of MS medium containing 500 mgL -1 vancomycin, 500 mgL -1 carbenicillin and supplemented with 2 mgL -1 trans-zeatin. The cultures were incubated at 25°C under a 16/8 h light/dark photoperiodic regime (40 kLux) and subcultured weekly on corresponding freshly prepared medium. Three weeks later, adventitious shoots emerged from the cut ends of the explants. The 1–2 cm lengths were transferred to fresh medium without zeatin. The regenerated shoots were able to produce roots on the same media. The putative transformed T0 plantlets were transferred to pots and grown to maturity under greenhouse condition. T0 plants were self-pollinated to get the next generation seeds. β-glucurodinase (gus) transformed tomato plantlets were obtained by co-cultivation of the hypocotyls explants with A. tumefaciens strain LBA4404 cells harboring the binary Ti vector pBI-102,24 and these plantlets were used as control plants for the subsequent experiments. PCR analysis. DNA was isolated from ectoine transgenic plants (T3) and from non-transformed plants (control) according to the method described by Rogers and Bendich 25 and subjected to PCR analysis using specific primer for the Ect C gene. The forward and reverse primers for the Ect C gene were: 5'-CAC TGG AGG ATC CAC ATG ATC GTT C-3' and 5'-CAG AAT AGA

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Figure 7. Peroxidase activity in leaves of control (MWT and SWT) and ectoine transgenic (MT, S1 and S2) tomato plant under salt stress. Values are means of three replicates.

Figure 8. Changes in malondialdehyde (MDA) concentration in leaves from ectoine transgenic and control tomato plants (WT) under salt stress.

ac leaf chamber (1-l) and an infrared gas analyzer (LI-6400; ©201 1L andeswith Bi os i enc e. GCT CCG GGT TAC AGC G-3' respectively. The reaction LI-COR, Lincoln, NE USA). The PAR on leaves was kept above Donotdi s t r i but e. mixture (20 μl) contained 10 ng DNA, 200 mM dNTPs, 1 mM 1,000 μmolm-2s-1 during the measurements. Measurements of of each primer, 0.5 units of Red Hot Taq polymerase (ABgene the fifth expanded leaf counting from the top leaf were taken Housse, UK) and 10-X Taq polymerase buffer (ABgene Housse, daily (at 11:00) after beginning salt treatment. Leaf temperature UK). The following profile was used for these reactions: 94°C/1 was maintained at 25°C. The LI-6400 infrared gas analyzer was min, followed by 30 cycles of 98°C/20 s, 68°C/1 min 30 s and used to measure stomatal conductance. There were ten replicates a final extension at 72°C/10 min. PCR products were separated per treatment. 13 by (2%) agarose gel electrophoresis and visualized with ethidium CO2 feeding. 13CO2 was administered through a mature leaf bromide. in the middle of the main stem 6 days after salt treatment in Northern blot analysis. Total RNA was isolated from trans- both wild-type and transgenic plants. The leaf was enclosed in a genic and control plants using the method of Chrigwin et al. transparent plastic bag and 350 μl of 13CO2 (99 13C atom %) was Northern hybridization was carried out using an RPN 3450 introduced from a cylinder. The leaf was allowed to assimilate Gene Image kit according to the manufacturer’s instructions 13C for 1 h at a PAR of more than 1,000 μmolm-2s-1. The plants (Amersham, Buckinghamshire, UK). were harvested 24 h after feeding and were cut into separate Salt-tolerance experiment. Control (gus transformed plants) parts: the 13CO2-fed leaf, non-fed leaves, stems including petioles tomato seedlings from the cultivar ‘Momotaro’ (MWT) and the and roots. Each plant part was weighed and dried in an open-air cultivar ‘Saturn’ (SWT) and three independent ectoine transgenic oven at 70°C for 3 days, then weighed and ground to powder in lines (T3) (MT, S1 and S2) derived from the two cultivars were a vibrating sample mill (TI-100; Heiko, Fukushima, Japan) for used. Seedlings were transferred into 3-l plastic pots containing measurement of 13C abundance. 13 a mixture of granite regosol, peat moss and perlite (2:1:1, v:v:v), C analysis. 13C abundance in the ground plant material watered daily with 400 ml of 10% Hoagland’s solution and the was determined with a mass spectrometer (Delta plus; Finnigan, soil water tension was maintained at ≤60 kP. At 30 days after San Jose, CA USA). The excess percentage of 13C atoms in the planting, the plants were subjected to salt stress by the addition sample was calculated as the difference in 13C atom percentage of 100, 200 or 300 mM NaCl to the daily supply of Hoagland’s between the sample and unlabelled plant samples. The amount solution for one week. The temperature in the greenhouse was of labeled carbon (A) in the plant sample was calculated using 28°C and the photosynthetically active radiation (PAR) was the following equation: A = 13C atom % excess in the sample/100 2,743 μmolm-2s-1. There were ten replicates per treatment. x amount of C in the plant part. The amount of total C in the Measurement of photosynthetic rate. The photosynthetic rate sample was determined by an element analyzer (EA1110; CE was measured with a portable photosynthesis system equipped Instrument Co., Ltd., Tokyo, Japan). The 13C partitioning rate


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(%) was calculated as 13C amount in the plant part/13C amount of absorption at 470 nm due to guaiacol oxidation. Activity was in the whole plant x 100. assayed for 1 min for each reaction solution (3 ml final volume), Determination of leaf water relations. Leaf water potential which comprised 100 mM potassium phosphate buffer (pH 7.0), (ψw) was measured by using a pressure chamber (Daiki-Rika 20 mM guaiacol, 10 mM H2O2 and 50 μl crude extract.27 Instruments, Tokyo, Japan) one week after starting salt treatDetermination of malondialdehyde concentration. ment. Following measurement of ψw the leaf samples were frozen Malondialdehyde (MDA) concentration was determined by in liquid nitrogen and stored at -20°C. Leaf tissues were thawed using the thiobarbituric acid (TBA) reaction as described by Fu and centrifuged at 1,200x g for 25 min at 4°C to extract the cell and Huang.28 Fresh leaves were homogenized in 5 ml of 0.1% sap. The osmotic potential (ψs) of the cell sap was measured using trichloroacetic acid (TCA). The homogenate was centrifuged at a vapor pressure osmometer (model 5,500; Wescor, Logan, UT 10,000x g for 5 min. One milliliter of the supernatant was added USA). Turgor potential was calculated by subtracting ψs from ψw. to 4 ml of 20% TCA containing 0.5% TBA. The mixture was Osmotic adjustment (OA) was calculated as the difference in ψs heated at 95°C for 30 min, quickly cooled on ice, and then cenbetween treated (salinized) and control plants. trifuged at 10,000x g for 10 min. Absorbance of the supernatant Measurement of plant dry weight. Plants were harvested one was read at 532 and 600 nm. After subtracting the non-specific week after starting the salt treatment. The harvested plants were absorbance at 600 nm, the MDA concentration was calculated dried at 80°C in an air-forced draught oven for more than three using an extinction coefficient of (155 mM-1cm-1).29 days, and then weighed. Statistical analysis. Statistical analysis was performed using Ectoine determination. Methanol-extracted ectoine was Analyze-it software (Analyze-it, Leeds, UK) in accordance with purified by ion-exchange chromatography17 and analyzed by 1H the method of Maxwell and Delany.30 nuclear magnetic resonance (NMR) spectroscopy using a JEOLAcknowledgments GSX 500 NMR instrument. The amount of ectoine present was calculated by comparing the peak area of ectoine with that of the We would like to thank Dr. K. Yoshida and Dr. A. Shimyo of internal standard (Formate). the Graduate School of Biological Science, Nara Institute of Determination of peroxidase activity. Isozymes were extracted Science and Technology, Japan, for providing the ectoine biofrom leaf tissue isolated from the transgenic tomato lines MT, S1 synthetic genes. Also, we would like to thank Mr. H. Fujitaka and S2, as well as from the control plants (MWT and SWT) sub- of the Instruments for Chemical Analysis Center, Hiroshima jected to salt stress. Leaf tissue (400 mg) was ground in 21ml ofde for ©2 01 L an sUniversity, Bi os c i enc e.his help with the NMR measurements of ectoine. extraction buffer (0.1% (w/v) Tris-citric acid, pH 7.5; 1% (w/v) This research was supported by a Grant-in-Aid for Scientific Donotdi s t r i but e. polyvinyl pyrolidone (PVP); 0.1% (w/v) ascorbic acid and 0.1% Research from the Japan Society for the Promotion of Science (w/v) cysteine) and centrifuged at 5,333x g (JS-5.2 rotor) at 4°C (No. 02512) of the Ministry of Education, Culture, Sports, for 5 min. Peroxidase activity was measured following the change Science and Technology, Japan. References 1. Epstein E, Norlyn JD, Rush DW, Kingsbury RW, Kelley DB, Cunningham GA, et al. Saline culture of crops: A genetic approach. Science 1980; 210:399-405. 2. Cuartero J, Yeo AR, Flowers TJ. Selection of donors for salt-tolerance in tomato using physiological traits. New Phytol 1992; 121:63-96. 3. Kathuria H, Giri J, Nataraja KN, Murata N, Udayakumar M, Tyagi AK. Glycinebetaine-induced water-stress tolerance in codA-expressing transgenic indica rice is associated with upregulation of several stress responsive genes. Plant Biotech J 2009; 7:512-26. 4. Maheswari M, Varalaxmi Y, Vijayalakshmi A, Yadav SK, Sharmila P, Venkateswarlu B, et al. Metabolic engineering using mtlD gene enhances tolerance to water deficit and salinity in sorghum. Biol Plant 2010; 54:647-52. 5. Kishor PBK, Hong Z, Miao GH, Hu CAA, Verma DPS. Overexpression of Δ1-pyrrolin-5-carboxylate synthase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 1995; 108:1387-94. 6. Sheveleva E, Chamara W, Bohnert HJ, Jensen RG. Increased salt and drought tolerance by D-ononitol production in transgenic Nicotiana tobacum L. Plant Physiol 1997; 115:1211-9. 7. 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Zhang XY, Liang C, Wang GP, Luo Y, Wang W. The protection of wheat plasma membrane under cold stress by glycine betaine overproduction. Biol Plant 2010; 54:83-8. 10. Moghaieb REA, Tanaka N, Saneoka H, Hussein HA, Yousef SS, Ewada MA, et al. Expression of betaine aldehyde dehydrogenase gene in transgenic tomato hairy roots leads to the accumulation of glycine betaine and contributes to the maintenance of osmotic potential under salt stress. Soil Science and Plant Nutrition 2000; 46:873-83. 11. Pilon-Smits EAH, Ebskamp MJM, Jeuken MJW, Weisbeek PJ, Smeekens SCM. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol 1995; 107:125-30. 12. Nakayama H, Yoshida K, Ono H, Murooka Y, Shimyo A. Ectoine, the compatible solute of Halomonas elongata conferes hyperosmotic tolerance in cultured tobacco cells. Plant Physiol 2000; 122:1239-48. 13. Moghaieb REA, Tanaka N, Saneoka H, Murooka Y, Ono H, Morikawa H, et al. Characterization of salt tolerance in ectoine-transformed tobacco plants (Nicotiana tabaccum): photosynthesis, osmotic adjustment and nitrogen portioning. Plant Cell Environ 2006; 29:173-82. 14. Suwa R, Nguyen NT, Saneoka H, Moghaieb REA, Fujita K. Effect of salinity stress on photosynthesis and vegetative sink in tobacco plants. Soil Science and Plant Nutrition 2006; 52:243-50. 15. Koch KE. Carbohydrate-modulated gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 1996; 14-509.

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16. Suwa R, Fujimaki S, Suzui N, Kawachi N, Ishii S, Sakamoto K, et al. Use of positron-emitting tracer imaging system for measuring the effect of salinity on temporal and spatial distribution of 11C tracer and coupling between source and sink organs. Plant Sci 2008; 175:210-6. 17. Kalashnikov JE, Balakhnina TI, Zakrzhevsky DA. Effect of soil hypoxia on activation of oxygen and the system of protection from oxidative destruction on roots and leaves of Hordeum vulgare. Fiziol Rast Russian Journal Plant Physiology 1994; 41:583-8. 18. Yordanova RY, Christov KN, Popova LP. Antioxidative enzymes in barley plants subjected to soil flooding. Environ Exp Bot 2004; 51:93-101. 19. Fadzilla NM, Finch RP, Burdon RH. Salinity, oxidative stress and antioxidant responses in shoot cultures of rice. J Exp Bot 1997; 48:325-31. 20. Marschner H. Part I. Nutritional physiology. In: Marschner H, ed. Mineral Nutrition of Higher Plants. London: Academic Press, 1995:18-30. 21. Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 2002; 7:405-10. 22. Shakirova FM. Role of hormonal system in the manifestation of growth romoting and antistress action of salicylic acid. In: Hayat S, AhMDA A, Salicylic Acid—A Plant Hormone, Springer 2007; 69-89. 23. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarium 1962; 15:473-97. 24. Jefferson RA, Kavanagh TA, Bevan MW. GUS fusion. β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 1987; 6:3901-7.

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25. Rogers SO, Bendich AJ. Extraction of DNA from milligram amounts of fresh herbarium and mummified plant tissues. Plant Mol Biol 1985; 5:69-76. 26. Chrigwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonulease. Biochemistry 1979; 18:5294-9. 27. Polle A, Otter T, Seifert F. Apoplastic peroxidases and lignification in needles of Norway spruce Picea abies L. Plant Physiol 1994; 106:53-60.

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