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Expression of ectoine biosynthetic genes in tobacco plants (Nicotiana tabaccum) leads to the maintenance of osmotic potential under salt stress

R.E.A. Moghaieb1,2, N. Tanaka3, Hirofumi Saneoka1 and Kounosuke Fujita1

1Department of Environmental Dynamics and Management, Graduate School of Biosphere Sciences, Hiroshima University,
Higashi-Hiroshima, 739-8528, Japan.E.Mail fujiko@hiroshima-u.ac.jp, E.Mail saneoka@hiroshima-u.ac.jp
2
Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt.E.Mail moghaieb@hiroshima-u.ac.jp.
3
Center for Gene Science, Hiroshima University, Higashi-Hiroshima, 739-8527, Japan. E.Mail ntana@hiroshima-u.ac.jp

Abstract

The ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) biosynthetic genes (ect ABC) from Halomonas elongata was introduced to tobacco plants using the Agrobacterium mediated gene delivery system. The stable integration of the ectoine genes was confirmed by Southern blotting analysis. Expression of these genes could be detected in the transgenic tobacco plants by Northern analysis. The accumulation of ectoine in the transgenic plants under the salt stress condition detected by 1H-NMR analysis was higher in the roots than in the leaves. The data suggest that the accumulation of ectoine and Na+ ion in transgenic tobacco plants improve tolerance to salt stress by maintenance the cell osmotic potential at the level that allows water uptake under salt stress.

Key words

Ectoine, osmotic potential, salt tolerance, transformation, tobacco plant

Introduction

Agricultural productivity is severely affected by soil salinity, and the damaging effects of salt accumulation in agricultural soils have influenced ancient and modern civilizations. The detrimental effects of salt on plants are a consequence of both a 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). Transgenic plants harboring genes for the biosynthesis of mannitol, proline, ononitol, trehalose, betaine and fructan have shown a significant improvement in water stress tolerance (Sheveleva et al. 1997, Romero et al. 1997, Moghaieb et al. 2000).

In the present study, we investigate the physiological aspects of salinity tolerance in transgenic tobacco plants expressing the three ectoine biosynthesis genes at a whole-plant level. The transgenic plants were able to grow, flower and produce fruits in the presence of 200 mM sodium chloride. The higher ectoine accumulation in the transgenic plant cell improves the salinity tolerance by maintaining the osmotic potential of the cells. To our knowledge, we are the first to explain the effects of ectoine on salt tolerance at a whole-plant level

Method

Plant material and transgenic plants

A. tumifaciens strain LBA4404 cells containing the binary Ti vector pBI101 Hm ect. ABC (Nakayama et al. 2000) were grown overnight in 30 ml of LB medium containing 50 μg/ml kanamycin sulfates at 28 C. Cotyledonary leaf explants of the N. tabaccum c.v petit havana were prepared from 21-day-old seedlings were immersed in the Agrobacterium suspension for 5 min., blotted on sterilized filter paper and placed onto a co-cultivation medium. The co-cultivation medium consisted of MS medium (Murashige and Skoog 1962) containing 100 μM acetosyringon and supplemented with 1 mg/l BA and 0.1 mg/l NAA. After co-cultivation for three days, the explants were transferred to above describe medium in addition to 500 mg/l vancomycin, 500 mg/l carbenicillin, and 50 mg/l kanamycin for selection. The cultures were maintained at 25C under a 16/8 h light/dark photoperiodic regime (70 mol m-2 s-1). Three weeks later, adventitious shoots emerged from the cut end of the explants. The regenerated shoots were able to produce roots on the same media. Rooted shoots were transplanted to soil and plant regenerated under greenhouse conditions.

Southern blotting analysis

Total genomic DNA was isolated from both transformed and wild type plants according to Rogers and Bendich (1985). Labeling of the probes, hybridization, and detection were carried out with theGene Image kit, (RPN 3540, Amersham, England).

Northern blot analysis

Total RNA was isolated from leave and root samples from both of the transgenic and control plants according to Chrigwin et al. 1979), and Northern hybridization was carried out with the Gene Image kit.

Salt tolerance experiment

For the salt tolerance experiments, wild type and three independent (T2) tansgenic lines were grown in plastic pots (3 L), each containing a mixture of granite regosol, peat moss, and perlite (2:1:1 v:v:v), irrigated daily with 400 mL of 1/10 Hoagland solution, and the soil water tension was maintained ≤ 60 k Pa. At 30 days after planting, the plants were subjected to salt stress by the addition of 100 and 200 mM NaCl to Hoagland solution for [1] week.

Determination of leaf water relations

Leaf water potential (ψw) was measured by using a pressure chamber (Daiki-Rika Instruments, Tokyo, Japan) at the end of treatment. Osmotic potential (ψs) of the cell sap was measured using a vapor pressure osmometer (model 5,500, Wescor, Logan, UT, USA). Turgor potential was calculated by subtracting the (ψs) from (ψw). Osmotic adjustment (OA) was calculated as the differences in (ψs) between salinized and control plants.

Na+, K+ analysis

Samples were extracted with 10 mL of 1 N HCl for 24 h at room temperature. Na+ and K+ concentrations of the extracts were determined using a flame photometer (Eiko Instruments Inc., Tokyo).

Ectoine extraction of and NMR analysis

Methanol-extracted ectoine was purified by ion-exchange chromatography according to Yang et al. (1995), and analyzed by 1H NMR with a JEOL-GSX 500 NMR instrument. The amount of ectoine was calculated by comparison of the peak area of ectoine with that of the internal standard (Formate).

Results and Discussion

In the present study the tobacco plant was transformed with the ectoine biosynthetic genes (ect ABC) by an Agrobacterium tumifaciens-mediated gene-delivery system. The stable integration of the ectoine biosynthetic genes was confirmed by Southern analysis (Fig. 1-a). Although it is generally known that the expression of multiple CaMV35S promoters is easily silenced in a transgenic plant cell, our data show that at least four CaMV35S promoters can be transcribed at the same time (Fig.1 b).

a

b

Fig.1 a: Southern blot hybridization of DNA prepared from transgenic tobacco plants.Lanes A and B: transgenic , WT: non-transgenic plants, b: Northern blotting analysis from leaves showing ect.A, ect.B and ect.C respectively. Lanes 1, 2 and 3 were transgenic lines 1, 2 and 3 respectively. WT: non-transgenic plants.

Furthermore, the fact that ectoine can only be detected in ectoine transgenic plants indicates that each mRNA derived from each of the ect genes was properly translated to each enzyme of the ectoine synthetic pathway. The accumulation of ectoine increased with increasing salt concentrations in the media up to approximately 50 mole g-1 FW (Table 1). The ectoine concentrations were consistently higher in roots than in leaves; a reverse trend, however, was true for the mRNA amount. These results suggest that although the ectoine gene is expressed at higher levels in leaves than roots, the ectoine synthesized in leaves may be translocated to the roots; consequently, ectoine concentrations increase more considerably in the roots.

Table 1. Effect of salinity on Na+ and K+ (m M g-1 FW) and ectoine (μ mole g-1 FW) concentrations in ectoine transgenic tobacco and control Plants (WT).

Genotype

NaCl
(mm)

Na +

K+

Ectoine concentration

Leaf

Root

Leaf

Root

Leaf

Root

WT

0

0.00

0.00

0.01

0.02

0.0

0.0

100

0.14

0.15

0.02

0.00

0.0

0.0

200

0.22

0.21

0.02

0.01

0.0

0.0

Line 1

0

0.00

0.00

0.02

0.02

8.5

9.4

100

0.19

0.19

0.02

0.02

31.2

45.9

200

0.25

0.22

0.02

0.01

33.4

50.1

Line 2

0

0.00

0.00

0.03

0.02

8.1

9.2

100

0.13

0.13

0.03

0.02

32.8

41.3

200

0.28

0.30

0.03

0.02

49.9

55.5

Line 3

0

0.00

0.00

0.02

0.02

8.8

9.5

100

0.21

0.21

0.02

0.02

24.4

38.4

200

0.26

0.29

0.03

0.03

26.7

52.3

Table2. Effect of salinity on water relations of leaves from ectoine transgenic tobacco plant and control (WT)

Genotypes

NaCl
(mM)

ψw
(MPa)

ψs
(MPa)

ψp
(MPa)

OA

WT

0

- 0.27 0.03

- 1.1 0.02

0.83 0.05

 

100

- 0.25 0.05

- 1.29 0.07

1.04 0.04

0.19

200

- 0.5 0.02

- 1.35 0.05

0.85 0.07

0.25

Line 1

0

- 0.25 0.03

- 0.93 0.05

0.68 0.01

 

100

- 0.32 0.01

- 1.15 0.07

0.83 0.05

0.22

200

- 0.35 0.04

- 1.39 0.03

1.04 0.02

0.46

Line 2

0

- 0.28 0.01

- 1.00 0.04

0.72 0.07

 

100

- 0.40 0.04

- 1.18 0.02

0.78 0.06

0.18

200

- 0.42 0.02

- 1.61 0.08

1.19 0.04

0.61

Line 3

0

- 0.24 0.01

- 0.99 0.08

0.75 0.02

 

100

- 0.40 0.03

- 1.30 0.03

0.90 0.05

0.31

200

- 0.40 0.06

- 1.43 0.06

1.03 0.09

0.44

It is well known that osmotic adjustment involves the net accumulation of solutes in a cell in response to salinity, and, consequently, the osmotic potential decreases, which in turn attracts water into the cells and enables the turgor to be maintained. In the present study, the difference in ψs and O.A seemed to be related to the accumulation of ectoine and Na+ (Table 2). It has been reported that positive ψp is required for cell elongation and stomatal opening (Neumann et al. 1988). The present data indicate that the ψp values of the ectoine transgenic lines increased with increasing salt concentrations. The increase in ψp values may be responsible for the promotion of transgenic plant growth under salt stress conditions. These results agree with our previous results regarding transformation of tomato plants with the betaine aldehyd dehydrogenase gene (Moghaieb et al. 2000).

Based on the accumulation of mRNA and ectoine, we can conclude that ectoine is synthesized in plant leaves and is translocated into roots, thus leading to maintenance of the osmotic potential of the cell at a level that allows water uptake.

This study indicates that salt tolerance in plants can possibly be improved by metabolic engineering of the ectoine biosynthetic genes and should stimulate many more studies regarding the molecular and physiological mechanism of this important compatible solute in plant cells under salinity stress.

References

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Moghaieb REA, Tanaka N, Saneoka H, Hussein HA, Yousef SS, Ewada MA, Aly MAM and Fujita K (2000). 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 46, 873-883.

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Sheveleva E, Chamara W, Bohnert HJ and Jensen RG (1997). Increased salt and drought tolerance by D-ononitol production in transgenic Nicotiana tobacum L. Plant Physiology 115, 1211-1219.

Yang WJ, Nadolsk-Orczyk A, Wood KV et al. (1995). Near-isogenic lines of maize differing for glycinebetaine. Plant Physiology 107, 621-6

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