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Keywords:

  • osmotic potential;
  • stomatal conductance;
  • transformation;
  • water transport

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) biosynthetic genes (ect. ABC) from Halomonas elongata were introduced to tobacco plants using an Agrobacterium-mediated gene delivery system. The genes for ectoine biosynthesis were integrated in a stable manner into the tobacco genome and the corresponding transcripts were expressed. The concentration of ectoine under salt-stress conditions was higher in the roots than in leaves. A close relationship was found between stomatal conductance and the amount of transported nitrogen, suggesting that water transport through the xylem in the stem and transpiration may be involved in nitrogen transport to leaves. The data indicate that the turgor values of the ectoine transgenic lines increased with increasing salt concentration. The data revealed two ways in which ectoine enhanced salinity tolerance of tobacco plants. First, ectoine improved the maintenance of root function so that water is taken up consistently and supplied to shoots under saline conditions. Second, ectoine enhanced the nitrogen supply to leaves by increasing transpiration and by protecting Rubisco proteins from deleterious effects of salt, thereby improving the rate of photosynthesis.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Agricultural productivity is affected strongly by soil salinity. Damaging effects of salt accumulation in agricultural soils have influenced ancient and modern civilizations. Detrimental effects of salt on plants are a consequence of a water deficit, which causes osmotic stress, and effects of excess sodium ions on important biochemical processes.

Salinity reduces nitrogen uptake, translocation of nitrogen from roots to shoots, and assimilation of nitrate in the leaves (Peuke et al. 1996). Baki et al. (2000) reported that although enzymatic activity of nitrate reductase was decreased under salt stress, this decrease was not a factor in impairment of plant growth by salt stress. Apparently, nitrate uptake by roots as well as xylem loading might be involved in reduction of the nitrate supply to leaves, which may depress photosynthesis under salinity.

Several investigators have shown that stem diameter fluctuates diurnally with changes in light and water status: it reflects the plant water status more directly than soil moisture status and other climatic factors (Simonneau et al. 1993). Simonneau et al. (1993) used the micromorphometric method to observe rapid changes in stem diameter, which were closely related to the water status throughout the day.

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). Accumulation of these compatible solutes has been suggested as a major mechanism underlying the adaptation or tolerance of plants to osmotic stress (Rhodes & Hanson 1993; Hayashi et al. 1997).

Ectoine is a common compatible solute in halophilic bacteria (Csonka & Epstein 1996). The role and activities of ectoine are of special interest because it can be synthesized de novo, in bacterial cells, and its synthesis in several Streptomyces strains as a response to increased salinity and elevated temperature has been described (Malin & Lapidot 1996). More information has been accumulated regarding ectoine activity in living cells. Exogenously provided ectoine can reverse growth inhibition caused by osmotic stress in Escherichia coli (Jebbar et al. 1992), Corynebacterium glutamicum (Farwick, Siewe & Kramer 1995), and the soil bacterium Rhizobium meliloti (Talibart et al. 1994).

An important feature of osmoprotectants is that their beneficial effects are generally not species-specific. Therefore, alien osmoprotectants can be introduced into plants to protect their new host (Rontein, Basset & Hanson 2002). Transgenic plants harbouring genes for biosynthesis of mannitol (Tarczynski, Jensen & Bohnert 1993), proline (Kishor et al. 1995), ononitol (Sheveleva et al. 1997), trehalose (Romero et al. 1997), betaine (Hayashi et al. 1997; Sakamoto, Nurata & Murata 1998; Moghaieb et al. 2000), and fructan (Pilon-Smits et al. 1995) have shown marked improvement in water stress tolerance.

The ectoine biosynthetic pathway in Halomonas elongata is synthesized in three steps. The first step is catalysed by 2,4-diaminobutyrate aminotransferase (DAT), which converts the aspartate β-semialdehyde (ASA) to l-2,4-diaminobutyric acid (DABA). The second step, the acetylation of DABA to Nγ-acetyl l-2,4- diaminobutyric acid (ADABA), is promoted by DABA acetyltransferase (DAA). In the final step, ectoine synthase (ES) catalyses the cyclic condensation of ADABA to ectoine (Ono et al. 1999).

The present study intended to transform tobacco plants using ectoine biosynthetic genes, and to evaluate the improvement in salt tolerance in terms of biomass production and its parameters: ectoine biosynthesis, photosynthesis, nitrogen uptake, nitrogen translocation from roots to shoots, and water status.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Transformation experiment

Transformation of tobacco plants with ectoine genes

Tobacco (Nicotiana tabaccum cv. petit Havana) seeds were germinated on Murashige and Skoog medium (MS; Murashige & Skoog 1962). Cotyledonary explants were excised from 21-day-old-seedlings and cut in half; they were used for ectoine transformation.

Agrobacterium tumefaciensstrain LBA4404 cells that harbour the binary Ti vector pBI101 Hm ect. ABC (Nakayama et al. 2000) were grown overnight in 30 mL of Luria-Bertani (LB) medium containing 50 µg mL−1 kanamycin sulphates, and then collected by centrifugation at 1120 g for 5 min. The pellet was re-suspended in MS medium containing 100 µm acetosyringon. Tobacco leaf disc explants were immersed in the bacterial suspension for 5 min. Then, the explants were blotted with sterilized filter paper and placed on a co-cultivation medium consisting of MS medium with  100 µm acetosyringon  and  supplemented  with  1 mg  L−1 benzylaminopurine (BA) and 0.1 mg L−1 1-naphthaleneacetic acid (NAA). They were then incubated at 25 °C under a 16/8 h light/dark photoperiod (70 µmol m−2 s−1).

After co-cultivation for 3 d, the explants were transferred to a selection medium consisting of MS medium containing 500 mg L−1 vancomycin, 500 mg L−1 carbenicillin, and 50 mg L−1 kanamycin and supplemented with 1 mg L−1 BA and 0.1 mg L−1 NAA. The cultures were maintained at 25 °C under a 16/8 h light/dark photoperiod (70 µmol m−2 s−1). Three weeks later, adventitious shoots emerged from the cut ends of the explants. The regenerated shoots produced roots on the same media. Rooted shoots were transplanted to soil and plants were regenerated under greenhouse conditions. T1 seeds were harvested, grown on MS medium containing 100 mg L−1 kanamycin, and then homozygous seeds were selected.

Southern blotting analysis

Total genomic DNA was isolated from both transformed and non-transformed plants according to a method described previously by Rogers & Bendich (1985). Restriction enzyme treatment of 10 µg of DNA, separation in 1% agarose gels, and blotting to a nylon membrane followed standard protocols (Sambrook, Fritsch & Maniatis 1989). Labelling of the probes, hybridization, and detection was carried out using an RPN 3540 Gene Image kit according to instructions provided by the manufacturer (Amersham, Little Chalfont, Bucks., UK).

Northern blotting analysis

Total RNA was isolated from leaf and root samples from both the transgenic and control plants according to the method of Chrigwin et al. (1979), and Northern hybridization was carried out using an RPN 3450 Gene Image kit according to the instructions provided by the manufacturer (Amersham).

Salt-tolerance experiment

Plant growing and salt treatments

For the salt tolerance experiments, wild type and three independent transgenic lines (T2) were used. The transgenic lines used here were selected out of 12 lines which showed both mRNA and increased levels of ectoine, based on growth rate equivalent to or greater than non-transgenic plants. Seedlings were transferred into 3 L plastic pots containing a mixture of granite regosol, peat moss, and perlite (2 : 1 : 1; v : v : v). Seedlings were irrigated daily with 400 mL of 1/10 Hoagland solution, and the soil water tension was maintained at ≤ 60 kPa. At 30 d after planting, the plants were subjected to three levels of salt stress with application of 0, 100 or 200 m m NaCl to the daily supply of Hoagland solution for 1 week. The greenhouse air temperatures in the day and night were 28 and 20 °C, respectively; photosynthetically active radiation (PAR) was above 1000 µmol m−2 s−1. Five replications were performed per treatment.

Measurement of stem diameter

Changes in the stem diameter of the transgenic line No. 3 and non-transgenic tobacco plants were recorded using a microdisplacement detector at 5 min intervals over 24 h after initiation of salt treatment (Imai, Iwao & Fujiwara 1990). The stem was passed through the inside of a Hofman pinch-cock and a piece of 10-mm diameter tygon tube was placed between the stem and the displacement sensor; the pinch-cock screw was adjusted to hold the stem, the displacement sensor and the tygon tube in tandem. Sensors were connected to a computerized data acquisition system (NEC; Sanei Kogo Co., Ltd, Tokyo, Japan). Based on our control runs using a glass rod (12 mm diameter), the measurement sensitivity of the diameter was within ± 2 µm. Control measurements were run from the same power source and connected to the same data logger. Micro-placement sensors were fastened to the stem, and the diameters were recorded over the same period. All measurements were repeated three times.

Measurement of photosynthetic rate (Po)

The photosynthetic rate was measured using a portable photosynthesis system equipped with a leaf chamber and an infrared gas analyser (Model LI-6400; Li-Cor Inc., Lincoln, NE, USA). PAR on leaves was kept above 1000 µmol m−2 s−1 during measurements. Measurements of the fifth expanded leaf counted from the uppermost leaf were performed daily (at about 1100 h.) after the beginning of NaCl treatment. Leaf temperature was controlled at 25 °C. All measurements were repeated five times. The LI-6400 was also used to measure stomatal conductance and leaf internal CO2 concentration.

15N feeding

Six days after salinity treatment, tobacco roots were dipped into a solution of NH415NO3 (10 atom% excess, 100 p.p.m. N) in a 20 L container for 24 h. The plants were harvested and separated into leaves, stems, and roots. Plant materials were freeze dried for 72 h and dry weight was determined. The samples were ground to a fine powder with vibrating sample mill (Model Tl-100; Heiko Co., Ltd, Iwaki, Fukushima, Japan) for 15N determination. The 15N abundance in the powdered plant materials was determined with a mass spectrometer (model Delta plus; Finnigan Co. San Jose, CA, USA). The 15N atom% excess in the plant samples was calculated as the difference in the 15N atom% between the samples and unlabelled plant samples. The amount of 15N (A) in the plant sample was calculated according to the following equation shown below.

  • A = (15N abundance in the sample/100) × total N amount in the sample.

The amount of total N was determined by the element analyser (EA1110; CE Instrument Co., Ltd, Tokyo, Japan) facilitated in the mass spectrometer.

Determination of leaf water relationships

The leaf water potential (ψw) of the same leaf used for measurement of Po was measured using a pressure chamber (Daiki Rika Kogyo Co. Ltd, Tokyo, Japan) after 1 week of salt treatment. After measurement of ψw, the leaf samples were frozen in liquid nitrogen and stored at −20 °C. Leaf tissues were thawed and centrifuged at 1200 g for 25 min at 4 °C to extract the cell sap. The osmotic potential (ψs) of the cell sap was measured using a vapour pressure osmometer (model 5500; Wescor Inc., Logan, UT, USA). The turgor potential (ψp) was calculated by subtracting the (ψs) from (ψw). Osmotic adjustment (OA) was calculated as the difference in ψs between salt-treated and untreated plants.

Measurements of plant dry weight

Plants were harvested 1 week after initiation of the salt treatment. The harvested plants were separated into leaves, stems and roots, which were dried individually at 80 °C in an air-forced draught oven for more than 3 d, then weighed.

Na+, K+ and ectoine analysis

The freeze-dried samples of the same leaf with Po measurement were ground into a fine powder as described above. 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, Japan).

Methanol-extracted ectoine was purified using ion-exchange chromatography (Yang et al. 1995) and analysed by 1H NMR with a JEOL-GSX 500 NMR instrument. The amount of ectoine was calculated by comparing the peak area of ectoine with that of the internal standard (formate).

Statistical analysis

Statistical analysis was performed using Analyze-it (Analyze-it Software Ltd, Leeds, UK) according to Maxwell & Delaney (1989).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Transformation and plant biomass

The present study transformed tobacco leaf disc explants with  the  ectoine  biosynthetic  genes  (ect.  ABC)  using  an A. tumefaciens-mediated gene-delivery system. The presence of transgenes in the regenerated plants was confirmed using Southern blotting analysis. Southern blotting analysis indicated that the transgenic plant yielded 1–2 bands that were hybridized specifically with the ect. B gene (Fig. 1). Northern blot analysis showed that the transcripts that hybridized specifically to a probe were derived from the ectoine biosynthesis genes (ect. A, ect. B and ect. C) and accumulated in the transgenic plants, but not in the non-transgenic plants. The ect genes were more strongly expressed in leaves than in roots of the transgenic plants under saline conditions (Fig. 2).

image

Figure 1. Southern blot hybridization of DNA prepared from transgenic tobacco plants, transformed by the ectoine biosynthetic genes. DNA (10 µg) was cut by restriction enzymes Bg1 II (Lane A) and Xho I (Lane B), separated on 1% agarose, transferred to membranes and hybridized to a labelled probe (ect. B). WT, non-transgenic plant.

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image

Figure 2. Analysis of ectoine gene expression by RNA blot analysis. Total RNA was isolated from leaves from both the transgenic and nontransgenic tobacco plants. Equal amounts of RNA (30 µg) were blotted onto nylon membrane and hybridized with ectoine biosynthesis gene (ect. A,B,C) probes. Ribosomal RNA (rRNA) visualized by staining with ethidium bromide is shown as a control. Northern blotting analysis from leaves and roots, respectively, show the expression of ect. A, ect. B and ect. C gene. Lanes 1, 2 and 3 (L1, L2 and L3) were transgenic lines 1, 2 and 3, respectively. WT, non-transgenic plants.

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The ectoine transgenic tobacco lines (T2) as well as the non-transgenic plants were subjected to gradual increases in NaCl concentrations (0, 100 and 200 m m) for 1 week. The whole-plant dry weight decreased with increasing NaCl concentrations in the cultural medium (Table 1). The reduction was more remarkable in the non-transgenic than in the transgenic plants. Transgenic line 3 showed the highest dry weight, followed by line 2 and then line 1, at the same salt concentrations.

Table 1.  Effect of salinity on plant biomass in ectoine transgenic (T2 of lines 1–3) and non-transgenic (wild type) tobacco plants
GenotypesNaCl treatments (m m)Dry weight (g plant−1)Whole plant relative DW (%)
LeavesStemsRootsWhole plant
  1. Seedlings were planted into soil pots for 30 d, and then exposed to different NaCl treatments for 1 week. Values are means ± standard deviations of five replicates.

Wild type  02.4 ± 0.21.0 ± 0.10.3 ± 0.013.7100
1001.8 ± 0.10.9 ± 0.30.3 ± 0.032.9 79
2001.7 ± 0.10.6 ± 0.10.2 ± 0.012.4 64
Line 1  01.5 ± 0.11.1 ± 0.10.2 ± 0.022.8100
1001.3 ± 0.11.1 ± 0.20.1 ± 0.032.5 89
2001.2 ± 0.20.9 ± 0.10.1 ± 0.012.1 76
Line 2  01.8 ± 0.11.3 ± 0.30.2 ± 0.023.4100
1001.7 ± 0.21.2 ± 0.10.2 ± 0.013.1 92
2001.3 ± 0.21.2 ± 0.30.1 ± 0.012.6 78
Line 3  02.4 ± 0.11.8 ± 0.10.3 ± 0.034.5100
1002.0 ± 0.11.7 ± 0.10.2 ± 0.064.0 88
2002.0 ± 0.31.4 ± 0.10.2 ± 0.053.7 81

Stem diameter

Our data indicate that the stem diameter decreased rapidly after light exposure, but increased at night (Fig. 3). The decrease in stem diameter was more remarkable at 200 m m NaCl than in the control; the decrease was more pronounced in non-transgenic than in transgenic plants. The recovery of decreased stem diameter after turning off the light in the evening was also impaired by NaCl treatment; this impairment was more remarkable in non-transgenic than in transgenic plants.

image

Figure 3. Changes in stem diameter of non-transgenic (wild type) and transgenic (T2 of line 3) tobacco plants under salt stress. Seedlings were planted into soil pots for 30 d, and then exposed to 0 or 200 m m NaCl. Stem diameters were recorded using a microdisplacement detector at 5 min intervals over 24 h after initiation of salt treatment.

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Photosynthesis

The photosynthetic rate decreased after 1 d of salt treatment; the decrease was more pronounced with increasing NaCl concentrations (Fig. 4). The magnitude of the reduction of photosynthetic rate by NaCl was more conspicuous in the non-transgenic plants than in the transgenic plants. Among the transgenic plants, the reduction was less in line 2 than in the others. A similar tendency was observed for stomatal conductance, with the non-transgenic being impaired more by NaCl than the transgenic plants (Fig. 4). The reduction of leaf internal CO2 concentration induced by NaCl application was less in transgenic plants compared with non-transgenic plants (Fig. 4).

image

Figure 4. Effect of salinity on apparent photosynthetic rate, stomatal conductance and intercellular CO2 concentration in leaves of the transgenic (T2 of lines 1–3) and non-transgenic tobacco plants (wild type). Seedlings were planted into soil pots for 30 d, and then exposed to of the different NaCl treatments. The parameters were measured daily in the fifth leaf from the uppermost leaf. Values are means of five replicates.

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Leaf water relations

The ψs of the salt-treated plants decreased with increasing NaCl concentrations (Table 2). That decrease was more pronounced in line 2. The osmotic adjustment (OA) value increased with increasing NaCl concentrations; the increase was greater in transgenic plants than in non-transgenic plants, especially at 200 m m NaCl treatment. The ψp values increased with increasing salt concentration in transgenic plants but not in non-transgenic plants.

Table 2.  Effect of salinity on water relations in leaves of ectoine transgenic (T2 of lines 1–3) and non-transgenic (wild type) tobacco plants
GenotypesNaCl treatments (m m)ψw (MPa)ψs (MPa)ψp (MPa)OA
  1. Seedlings were planted into soil pots for 30 d, and then exposed to different NaCl treatments. On the seventh 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 the Materials and methods. Values are means of three replicates.

Wild type  0−0.27−1.100.83 
100−0.25−1.291.040.19
200−0.50−1.350.850.25
Line 1  0−0.25−0.930.68 
100−0.32−1.150.830.22
200−0.35−1.391.040.46
Line 2  0−0.28−1.000.72 
100−0.40−1.180.780.18
200−0.42−1.611.190.61
Line 3  0−0.24−0.990.75 
100−0.40−1.300.900.31
200−0.40−1.431.030.44

Concentrations of Na+, K+ and ectoine

Na+ concentration in the leaves and roots increased with the increasing NaCl concentration in the treatment media (Table 3); the increase was greater in the transgenic than in the non-transgenic plants. The transgenic plants accumulated higher Na+ concentrations in the roots than in the leaves except for line 1 at 200 m m NaCl. Among the transgenic plants, lines 2 and 3 exhibited higher Na+ concentrations in both roots and leaves at 200 m m treatment; at 100 m m treatment, however, line 2 exhibited lower Na+ concentrations compared with others.

Table 3.  Effect of salinity on concentrations (µmol g−1 fresh weight) of Na+, K+, and ectoine in leaves and roots of ectoine transgenic (T2 of lines 1–3) and non-transgenic (wild type) tobacco plants
GenotypesNaCl treatments (m m)Na+K+Ectoine
RootsLeavesRootsLeavesRootsLeaves
  1. Seedlings were planted into soil pots for 30 d, and then exposed to different NaCl treatments for 1 week. Concentration of Na+, K+ and ectoine were measured in the fifth expanded leaf counted from the uppermost leaf. Values are means ± standard deviations of five replicates.

Wild type  0  8.6  6.2544200
100 85 78407400
200112121306300
Line 1  0 10.9  8.16876 9.4 ± 0.2 8.5 ± 0.1
100121111727445.9 ± 0.731.2 ± 0.1
200128151426650.1 ± 0.533.4 ± 0.4
Line 2  0 11.3  7.36278 9.2 ± 0.3 8.1 ± 0.3
100 94 75729241.3 ± 0.432.8 ± 0.2
200165162789255.5 ± 0.949.9 ± 0.1
Line 3  0 13.4  8.86262 9.5 ± 0.2 8.8 ± 0.2
100133121787038.4 ± 0.624.4 ± 0.1
200173163748452.3 ± 0.426.7 ± 0.5

The data indicated that the K+ concentration was slightly increased by NaCl treatment in lines 2 and 3 and in leaves of the non-transgenic plants. In contrast, it was decreased in line 2 and roots of the non-transgenic plants (Table 3).

Ectoine accumulation increased with increasing salt concentrations in the media up to approximately 50 µmol g−1 FW (Table 3). Ectoine concentrations were consistently higher in roots than in leaves. There were significant differences in root ectoine concentration among the examined transgenic lines at both NaCl treatments; line 1 and 2 accumulated higher concentrations at 100 and 200 m m NaCl, respectively. A positive relationship was found between the ectoine concentration in roots of transgenic plants and the increase in relative plant biomass of transgenic plants compared with non-transgenic plants at the same NaCl treatment (Fig. 5).

image

Figure 5. The relationship between ectoine concentration in roots (Table 2) and the increase in relative dry weight, which was the difference between the relative dry weight of transgenic plants and non-transgenic plants at the same NaCl treatment (Table 1).

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15N uptake and export

The 15N atom percentage excess was higher in leaves and stems in the transformed than in the non-transformed plants, but it did not differ between roots of transformed and non-transformed plants at 0 m m NaCl (Table 4). It decreased in stems of all the plants in response to saline treatment, but tended to increase in the roots.

Table 4.  Effect of salinity on 15N atom% excess in various plant parts of ectoine transgenic (T2 of lines 2 and 3) and non-transgenic (wild type) tobacco plants
GenotypesPlant partsNaCl treatments (m m)
0100200
  1. Seedlings were planted into soil pots for 30 d and then exposed to different NaCl treatments. Six days after salinity treatment, tobacco roots were dipped into a solution of NH415NO3 (10 atom% excess, 100 p.p.m. N) for 24 h. Values are means ± standard deviations of five replicates. 15N atom% excess was calculated as described in the Materials and Methods.

Wild typeLeaves0.13 ± 0.010.11 ± 0.030.13 ± 0.02
Stems0.27 ± 0.080.17 ± 0.030.13 ± 0.09
Roots0.15 ± 0.050.33 ± 0.030.27 ± 0.17
Line 2Leaves0.25 ± 0.040.24 ± 0.030.09 ± 0.01
Stems0.34 ± 0.060.24 ± 0.080.16 ± 0.02
Roots0.16 ± 0.030.27 ± 0.070.15 ± 0.01
Line 3Leaves0.23 ± 0.020.11 ± 0.020.13 ± 0.04
Stems0.37 ± 0.030.16 ± 0.050.15 ± 0.07
Roots0.15 ± 0.050.17 ± 0.040.15 ± 0.04

In the absence of salt, N uptake in the transformed plants was about twice that in the non-transformed plants throughout the experimental period (Fig. 6). It decreased in all the plants under saline conditions: that decrease was less pronounced in the non-transgenic than in the transgenic plants. In contrast, the 15N export rate from roots to shoots was higher in the transgenic than in the non-transgenic plants. Changes in 15N uptake and export by transformation and salinity resulted in a tendency to increase the amount of 15N in leaves of transgenic compared with non-transgenic plants under salt treatment.

image

Figure 6. The effect of salinity on the amount of 15N partitioning to different plant parts of the transgenic (T2 of lines 2 and 3) and non-transgenic (wild type) tobacco plants. Seedlings were planted into soil pots for 30 d, and then exposed to of the different NaCl treatments. Six days after salinity treatment, tobacco roots were dipped into a solution of NH415NO3 (10 atom% excess, 100 p.p.m. N) for 24 h. Values are means of five replicates.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Expression of multiple CaMV35S promoters is easily silenced in transgenic plant cells, but our data show that at least four CaMV35S promoters can be transcribed simultaneously. Furthermore, ectoine was detected only in ectoine transgenic plants, indicating that each mRNA derived from each of the ect genes was translated properly to each enzyme of the ectoine synthetic pathway. Accumulation of ectoine increased with increasing salt concentration in the media [to approximately 50 µmol g−1 FW (Table 3)]. This restriction might result from post-transcriptional regulation of the ectoine biosynthetic genes under salt stress. These results are consistent with our previous findings concerning post-transcriptional regulation of the BADH gene under salt stress (Moghaieb et al. 2000). These results are also consistent with the findings of Hayashi et al. (1997) who reported that when Arabidopsis thaliana was transformed with the cod A gene under the control of the CaMVP 35 S, the transgenic plant was able to accumulate betaine. The rates of ectoine accumulation in the present study are much higher than those for the transgenic tobacco cell lines (70 nmol g−1 FW) previously reported by Nakayama et al. (2000), and are equivalent to or higher than the glycine betaine concentrations in maize (17 µmol g−1 FW) (Yang et al. 1995). Ectoine concentrations were consistently higher in roots than in leaves (Table 3). However, the opposite trend was observed for the mRNA amount (Fig. 2). 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.

The data in the present study showed a positive relationship between ectoine concentration in roots and plant growth (Fig. 5). The depression of whole plant dry weight by application of NaCl was to a less extent in the transgenic tobacco plant than wild one (Table 1), in parallel to this change, ectoine concentration in leaves and/or roots tended to increase except that the increase in ectoine concentration between 100 and 200 m m in line 1 was only slight with unknown reasons (Table 3). These results indicate that ectoine synthesis may play a role on protection of plant growth from salinity injury.

The data indicate that depression of the photosynthetic rate by salinity is caused by a reduction in both the stomatal conductance and carboxylation activity (Fig. 4); the former is particularly more impaired than the latter, as shown by the greater reduction of stomatal conductance compared with leaf internal CO2 concentration. Several recent papers have shown that the effects of salinity on photosynthesis are largely stomatal, but the increase in Na+ and Cl concentrations in the leaf, as leaves age, induces the non-stomatal inhibition. Ectoine therefore might protect leaves against high internal Na+, or Cl concentrations and play a role in water relations. A close correlation between the photosynthetic rate and the whole-plant weight was observed under different environmental conditions, implying that the impairment of plant biomass production by salt is caused largely by photosynthetic activity. Considering the differences in leaf weight, the estimated whole-plant photosynthetic rate correlates more with the whole-plant weight. The reduction in photosynthetic rate was more remarkable in non-transgenic than in transgenic plants.

The ability of osmoprotectants (e.g. proline and betaine) to overcome the inhibitory effect of osmotic stress in bacteria has conventionally been attributed to the special interactions of proline and betaine with proteins, which protect them from denaturation in the presence of high concentrations of electrolytes (Csonka 1989). In support of this mechanism, it has been shown that ectoine stabilizes proteins upon freezing and at high temperatures (Galinski 1993). These results are consistent with those reported by Yu & Nagaoka (2004), who found that ectoine protected the protein from salt injury by maintaining the protein hydration structure, thereby slowing water diffusion around the protein. The present results indicate that transgenic plants are more tolerant of salinity stress than non-transgenic plants because of the protection of photosynthetic activity as well as the sink activity of roots and stems.

It has been reported that proline and betaine merely maintain cell turgor in high osmolarity media, being compatible with normal cellular functions at high intercellular concentration. The intercellular concentration of ectoine is as high as 158 m m in non-halophilic Streptomyces bacteria (Malin & Lapidot 1996), and as high as 2.25 m in the halophilic bacterium H. elongata (Wohlfarth, Severin & Galinski 1990), whereas betaine can be synthesized to 0.6 m in the Methanohalophilus strain Z7401 (Lai et al. 1991). At these high concentrations, proline, betaine and ectoine demonstrate pronounced destabilizing effects on DNA in vitro (Rees et al. 1993). Current data indicate that accumulation of ectoine in plant cells serves to maintain higher ψp values under salt stress. The increase in ψp values may be responsible for promotion of transgenic plant growth under salt-stress conditions. These findings agree with our previous results regarding transformation of tomato plants with the betaine aldehyde dehydrogenase gene (Moghaieb et al. 2000).

It has been well documented that plant water status is detectable by changes in the stem diameter recorded by the micromorphometric method (Simmoneau et al. 1993). Our data showing that stem diameter decreased rapidly after exposure to light, but increased at night (Fig. 3), support the findings of previous authors who assumed that these responses were caused mainly by changes in the stomata aperture (Simmoneau et al. 1993; Ito et al. 1999). Our current data indicate that the stem diameter decreases during daylight and that the decrease is promoted by NaCl treatment (Fig. 3). Moreover, the stomatal opening in transgenic plants was greater than that in non-transgenic plants, as evidenced by high stomatal conductance in the former (Fig. 4). These results suggest that when stomata open after the exposure to light, the loss of water by transpiration becomes greater than the supply of water by the roots, engendering stem shrinkage. The decrease in stem diameter was more remarkable at 200 m m NaCl than in the control (0 m m NaCl); the decrease was more pronounced in non-transgenic (wild type) than in transgenic plants (Fig. 3). Recovery of the decreased stem diameter after turning off the light in the evening was also impaired by NaCl treatment, and the impairment was more remarkable in non-transgenic than in transgenic plants. These results suggest that under high-salinity conditions, the changes in the stem diameter reflect the water supply from roots, which is associated with the ability of roots to take up water.

It has been reported that when the water supply increases, ammonia transport is also promoted through the xylem in barley stems, as determined by monitoring with a positron-emitting tracer imaging system (PETIS) (Kiyomiya et al. 2001). That study found that, particularly when transpiration was extremely low under dark conditions, transport of 13N was strongly impaired. Mori et al. (2000) reported that water transport is closely associated with the manipulation of stomatal opening by abscisic acid. That study found a clear relationship between water uptake and nitrogen uptake and upward transport in the plant shoot. We observed a close correlation between transpiration and 15N partitioning to leaves using soybean under different atmospheric CO2 concentrations and that under high CO2 concentration, 15N partitioning into leaves decreased significantly in comparison with ambient air condition (data not shown). The present study demonstrated a positive relationship between stomatal conductance and the amount of nitrogen transported at different NaCl concentration using non-transgenic and transgenic plants (Fig. 7). This fact suggests that water transport through the xylem in the stem is involved in the transport of nitrogen to leaves. When the stomatal conductance is high, more nitrogen seems to accumulate in the leaves of transgenic plants. This finding is supported by the existence of the influence of the transpiration stream on the xylem transport of malate; the influence of abscisic acid can not be excluded (Peuke et al. 1996). Nitrate loading in the root xylem is also putatively involved in nitrate transport from the roots to the leaves (Baki et al. 2000). However, it is hypothesized that reductions in transpiration by stomatal closure have nothing to do with the rate of transport of solute in the xylem, except for the situation of very low rates of transpiration as at night (Munns 2002).

image

Figure 7. The relationship between leaf stomatal conductance (at the sixth day in Fig. 4) and leaf 15N amount (Fig. 6) in several transgenic and non-transgenic tobacco plants.

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It remains to be determined why stomatal conductance is maintained at a higher level in transgenic than in non-transgenic plants under conditions of salt stress (Fig. 4). The present study revealed that ectoine plays an important role as an osmoticum in maintaining higher moisture content in leaves; in addition, open stomata under saline conditions lead to enhanced transpiration. Consequently, water transpiration through the stomata stimulates translocation of water through the xylem from roots. The water flow, which is regulated mostly by stomata opening, promotes nitrogen translocation.

Taken together, the results of the present study show that ectoine enhances the salinity tolerance of tobacco plants in two ways. First, ectoine improves the maintenance of root function so that water can be taken up consistently and supplied to shoots under saline conditions. Second, ectoine enhances the nitrogen supply to leaves by increasing transpiration and by protecting Rubisco proteins from deleterious effects of salt, thereby improving the rate of photosynthesis.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We would like to thank Dr K. Yoshida and Dr A. Shimyo of the Graduate School of Biological Science, Nara Institute of Science and Technology, Japan, for provision of the ectoine biosynthetic genes. We would also like to thank Mr H. Fujitaka of the Instrument for Chemical Analysis Center, Hiroshima University, for his help in the NMR measurements of ectoine. This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 02512) of the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  • Baki G., Abd El Siefritz K.F., Man H.M., Weiner H., Kaldenhoff R. & Kaiser W.M. (2000) Nitrate reductase in Zea mays L. under salinity. Plant, Cell and Environment 23, 515521.
  • Chrigwin J.M., Przybyla A.E., MacDonald R.J. & Rutter W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemical Journal 18, 52945299.
  • Csonka L.N. (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiology Reviews 53, 121147.
  • Csonka L.N. & Epstein W. (1996) Osmoregulation. In Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. F.C.Neidhardt), 2nd edn, pp. 12101223. ASM Press, Washington, DC, USA.
  • Farwick M., Siewe R.M. & Kramer R. (1995) Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum. Journal of Bacteriology 177, 46904695.
  • Galinski E.A. (1993) Compatible solutes of halophilic eubacteria: molecular principles, water–solute interaction, stress protection. Experientia 49, 487495.
  • Hayashi H., Alia Mustardy L., Deshnium P., Ido M. & Murata N. (1997) Transformation of Arabidopsis thaliana with the cod A gene for choline oxidase; accumulation of glycine betaine and enhanced tolerance to salt and cold stress. Plant Journal 12, 1331342.
  • Imai S., Iwao K. & Fujiwara T. (1990) Measurement of plant physiological information of vine tree and indexation of soil moisture control. (1) Analysis of stem diameter variation affected by environmental factors. Environmental Control Biology 28, 103108.
  • Ito J., Hasegawa S., Fujita K., Ogasawara S. & Fujiwara T. (1999) Effect of CO2 enrichment on fruit growth and quality in Japanese pear (Pyrus serotina reheder cv. Kosui). Soil Science and Plant Nutrition 45, 385393.
  • Jebbar M., Talibart R., Gloux K., Bernard T. & Blanco C. (1992) Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics. Journal of Bacteriology 174, 50275035.
  • Kishor P.B.K., Hong Z., Miao G.H., Hu C.A.A. & Verma D.P.S. (1995) Overexpression of Δ1-pyrrolin-5-carboxylate synthase increases proline production and confers osmotolerance in transgenic plants. Plant Physiology 108, 13871394.
  • Kiyomiya S., Nakanishi H., Uchida H., et al. (2001) Real time visualization of 13N-translocation under different environmental conditions using positron emitting tracer imaging system. Plant Physiology 125, 17431754.
  • Lai M.C., Sowers K.R., Robertson D.E., Roberts M.F. & Gunsalus R.P. (1991) Distribution of compatible solutes in the halophilic methanogenic archaebacteria. Journal of Bacteriology 173, 53525535.
  • Malin G. & Lapidot A. (1996) Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress. Journal of Bacteriology 178, 385395.
  • Maxwell S.E. & Delaney H.D. (1989) Designing Experiments and Analyzing Data. pp. 241260. Wadsworth, Belmont, CA, USA.
  • Moghaieb R.E.A., Tanaka N., Saneoka H., Hussein H.A., Yousef S.S., Ewada M.A., Aly M.A.M. & 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, 873883.
  • Mori S., Kiyomiya S., Nakanishi H., Ishioka N.S., Watanabe S., Osa A., Matsuhashi S., Hashimoto S., Sekine T. & Uchida (2000) Utilization of 15 O-water flow in tomato and rice in the light and dark using a positron emitting tracer imaging system (PETIS). Soil Science and Plant Nutrition 46, 975979.
  • Munns R. (2002) Comparative physiology of salt and water stress. Plant, Cell and Environment 25, 239250.
  • Murashige T. & Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15, 473379.
  • Nakayama H., Yoshida K., Ono H., Murooka Y. & Shimyo A. (2000) Ectoine, the compatible solute of Halomonas elongata confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiology 122, 12391248.
  • Ono H., Sawada K., Khunajakr N., Tao T., Yamamoto M., Hiramoto M., Shinmyo A., Takano M. & Murooka Y. (1999) Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacteria, Halomonas elongata. Journal of Bacteriology 181, 9199.
  • Peuke A.D., Glaab J., Kaiser W.M. & Jeschke W.D. (1996) The uptake and flow of C, N and ions between roots and shoots in Ricinus communis L. IV. Flow and metabolism of organic nitrogen and malate depending on nitrogen nutrition and salt treatment. Journal of Experimental Botany 47, 377385.
  • Pilon-Smits E.A.H., Ebskamp M.J.M., Jeuken M.J.W., Weisbeek P.J. & Smeekens S.C.M. (1995) Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiology 107, 125130.
  • Rees W.A., Yager T.D., Korte J. & Von Hippel P.H. (1993) Betaine can eliminate the base pair composition dependence of DNA melting. Biochemical Journal 32, 137144.
  • Rhodes D. & Hanson A.D. (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annual Review of Plant Molecular Biology 44, 357384.
  • Rogers S.O. & Bendich A.J. (1985) Extraction of DNA from milligram amounts of fresh herbarium and mummified plant tissues. Plant Molecular Biology 5, 6976.
  • Romero C., Belles J.M., Vaya J.L., Serrano V.R. & Culianez-Macia F.A. (1997) Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance. Planta 201, 293297.
  • Rontein D., Basset G. & Hanson A.D. (2002) Metabolic engineering of osmoprotectant accumulation in plants. Metabolic Engineering 4, 4956.
  • Sakamoto A., Murata A. & Murata N. (1998) Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold. Plant Molecular Biology 38, 10111019.
  • Sambrook J., Fritsch E.F. & Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • Sheveleva E., Chamara W., Bohnert H.J. & Jensen R.G. (1997) Increased salt and drought tolerance by d-ononitol production in transgenic Nicotiana tobacum L. Plant Physiology 115, 12111219.
  • Simonneau T., Habib R., Goutouly J.P. & Huguet J.G. (1993) Diurnal changes in stem diameter depend upon variations in water content: direct evidence in peach trees. Journal of Experimental Botany 44, 615621.
  • Talibart R., Jebbar M., Gouesbet G., Himdi-Kabbab S., Wroblewski H., Blanco C. & Bernard T. (1994) Osmoadaptation in rhizobia: ectoine-induced salt tolerance. Journal of Bacteriology 176, 52105217.
  • Tarczynski M.C., Jensen R.G. & Bohnert H.J. (1993) Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science 259, 508510.
  • Wohlfarth A., Severin J. & Galinski E.A. (1990) Heterotrophic halophilic eubacteria of the family Halomonadaceae. Journal of General Microbiology 136, 705712.
  • Yang W.J., Nadolsk-Orczyk A., Wood K.V., et al. (1995) Near-isogenic lines of maize differing for glycinebetaine. Plant Physiology 107, 621630.
  • Yu I. & Nagaoka M. (2004) Slowdown of water diffusion around protein in aqueous solution with ectoine. Chemical Physics Letters 388, 316321.