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Keywords:

  • Nicotiana tabacum;
  • chilling sensitivity;
  • glycerol-3-phosphate acyltransferase;
  • inflorescence;
  • reproductive stage

ABSTRACT

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

The cis-unsaturated molecular species of phosphatidylglycerol (PG) in chloroplasts have been implicated in the chilling sensitivity of plants. Homozygous lines of transgenic tobacco (Nicotiana tabacum) that overexpressed the cDNA for glycerol-3-phosphate acyltransferase, a key enzyme in the determination of the extent of cis-unsaturation of PG, were established from a chilling-sensitive squash (Cucurbita moschata). In transgenic plants, the proportion of saturated plus trans-monounsaturated molecular species of PG increased from 24 to 65%. However, this change did not affect the architecture of the chloroplasts. Chilling stress decreased the growth and biomass production of young seedlings of transgenic plants more severely than those of wild-type plants, and this observation suggests that the changes in the proportion of cis-unsaturated PG affected not only leaves but also developing plants. Chilling stress also damaged inflorescences. In particular, the abscission of flower buds and inflorescence meristems from transgenic plants occurred more frequently than that from wild-type plants. Thus, it is likely that decreases in the proportion of cis-unsaturated PG enhanced the sensitivity to chilling of reproductive organs.


INTRODUCTION

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

Low temperature is an important environmental stress that affects the growth and development of plants. In virtually all habitats, such stress causes significant loss of plant productivity (Graham & Patterson 1982). A close correlation exists between the chilling sensitivity of plants and the extent of cis-unsaturation of the fatty acids in the phosphatidylglycerol (PG) that is found in the thylakoid membranes of their chloroplasts (Murata et al. 1982; Murata 1983). In previous studies, we demonstrated that sensitivity of tobacco (Nicotiana tabacum) plants to low temperature could be modified genetically by altering the level of unsaturated fatty acids in PG (Murata et al. 1992). Such metabolic  manipulation  was  achieved  by  transformation  of tobacco plants with cDNA for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT), a nucleus-encoded chloroplast protein, since the substrate selectivity of this enzyme determines the extent of unsaturation of fatty acids in PG (Murata et al. 1992; Murata & Tasaka 1997). We found subsequently that unsaturation of fatty acids in membrane lipids accelerated the recovery of the photosynthetic machinery from photo-inhibition but did not affect the light-induced inactivation that occurred during photo-inhibition (Moon et al. 1995). In our previous studies, we focused on the chilling sensitivity of leaves at the vegetative stage of growth.

The present study was designed to investigate changes in the sensitivity to low temperature, caused by transformation with a cDNA for GPAT from squash (Cucurbita moschata; Murata et al. 1992), at the developing and reproductive stages of the life cycle of tobacco plants. We established homozygous lines of transgenic plants that overproduced GPAT from squash and then examined  the morphological and physiological consequences of the transformation. We found that genetic manipulation of the unsaturated fatty acids in PG altered the sensitivity to chilling of the reproductive organs and of developing leaves.

MATERIALS AND METHODS

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

Plant materials and culture conditions

Wild-type and transgenic tobacco plants were obtained and grown as described previously (Murata et al. 1992). Plants grown in pots were supplied with 0.1% (v/v) Hyponex (Hyponex Corporation, Marysville, OH, USA) on a regular basis.

Southern blotting analysis

Southern blotting analysis was performed with total DNA from leaves of mature plants. After digestion of the DNA with HindIII, EcoRI or SacI, fragments of DNA were fractionated, blotted onto a nylon membrane, and allowed to hybridize with an 847-bp fragment of squash cDNA for GPAT that had been obtained by digestion of λAT03 with HindIII and EcoRI (Ishizaki et al. 1988). Prehybridization, hybridization, washing and detection of signals were performed, according to the manufacturer's instructions, with an AlkPhos Direct Labelling and Detection System with CDP-Star® (Amersham Pharmacia Biotech, Uppsala, Sweden).

Analysis of fatty acid composition

Lipids were extracted from leaves as described by Bligh & Dyer (1959). PG was fractionated by ion-exchange column chromatography and then purified by thin-layer chromatography on silica gel as described previously (Murata et al. 1982). Purified PG was subjected to methanolysis and the resultant methyl esters were analysed by gas-liquid chromatography (Murata et al. 1982).

Growth under low-temperature conditions

Surface-sterilized seeds were sown on Murashige–Skoog medium that had been solidified with 0.5% (w/v) Gellum Gum (Wako, Osaka, Japan). Seeds were allowed to germinate and seedlings were grown aseptically at various temperatures (10, 15, 20 and 25 °C) with a 16 h light (0.15 mmol m−2 s−1)/8 h dark cycle in temperature-controlled growth cabinets. After 1 month, the plants were photographed, and weights of fresh and dry matter were determined.

Electron microscopy

Plants that had been grown for 3 months were incubated in darkness for 36 h to deplete chloroplasts of starch granules. Then small pieces were excised from fully developed mature leaves and fixed in a modified version of Karnovsky's fixative, in which cacodylate had been replaced by phosphate (Karnovsky 1965). After rinsing with 0.1 m sodium phosphate buffer (pH 7.2), samples were post-fixed in 1% (w/v) OsO4 for 1 h, dehydrated, and embedded in Epon 812 (Shell Chemical Co., San Francisco, CA, USA) as described previously (Ferjani et al. 2003). Thin sections were prepared from the samples with an ultramicrotome (Ultracut-T; Leica, Solms, Germany) and mounted on copper grids. They were then stained with uranyl acetate and lead citrate and examined with an electron microscope (JEM-100CX; JEOL, Tokyo, Japan).

RESULTS AND DISCUSSION

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

Establishment of homozygous lines of transgenic tobacco plants

In a previous study, we transformed tobacco plants with a binary plasmid, pSQ, in which a cDNA that encoded the mature form of squash GPAT had been fused to the cDNA for a chloroplast-targeting signal and placed under the control of the cauliflower mosaic virus 35S promoter (Murata et al. 1992). This transformation yielded plants that overexpressed the squash enzyme in their chloroplasts. In our previous studies, we examined heterozygous lines of transgenic tobacco plants (Murata et al. 1992; Moon et al. 1995) and, thus, for the present study, we attempted first to establish homozygous lines by repeated self-crossing, using kanamycin resistance as a marker. The T1 progeny of two independently transformed T0 plants (SQ7 and SQ8) gave a ratio of 3 : 1 for kanamycin resistance, indicating that the cDNA for GPAT had been integrated at a single locus. We selected T2 plants derived from individual T1 plants of the SQ7 and SQ8 lines by segregation analysis for antibiotic resistance in order to establish homozygous lines. The presence of GPAT cDNA in two sublines of each homozygous line (SQ7-05 and SQ7-11 for the SQ7 line; SQ8-13 and SQ8-15 for the SQ8 line), which had diverged at the T1 generation from each of the two independent lines, was confirmed by the polymerase chain reaction (data not shown) and these sublines were used for subsequent experiments.

The various transgenic plants exhibited no distinguishable differences from wild-type plants in terms of gross morphology under normal growth conditions. We performed Southern blotting analysis to obtain an estimate of the number of copies of the transgene for GPAT in the transgenic plants, using total DNA that had been isolated from leaves of T3 plants. Figure 1 shows that each respective set of sublines was identical in terms of the number of copies and pattern of integration of the transgene. The number of hybridization signals suggested that a single copy of the transgene had been integrated in the SQ7 lines, whereas the SQ8 lines might have incorporated two copies of the transgene at a single locus.

image

Figure 1. Southern blotting analysis of the number of copies of the GPAT transgene in T3 transgenic tobacco plants. Aliquots of DNA (20 µg) from wild-type (lanes 1, 6 and 11), SQ7-05 (lanes 2, 7 and 12), SQ7-11 (lanes 3, 8 and 13), SQ8-13 (lanes 4, 9 and 14) and SQ8-15 (lanes 5, 10 and 15) plants were digested with EcoRI (lanes 1–5), HindIII (lanes 6–10) and EcoRI plus SacI (lanes 11–14). After gel electrophoresis and blotting, the membrane was probed with an 847-bp fragment of GPAT cDNA. Lengths of fragments are shown in kb on the left.

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Changes in fatty acid composition of phosphatidylglycerol

We reported previously that overexpression of squash GPAT in the chloroplasts of transgenic tobacco plants led to increased levels of saturated fatty acids in the PG in chloroplasts without any significant changes in the relative levels of lipid classes in the chloroplast membranes (Murata et al. 1992; Moon et al. 1995). Table 1 shows that this biochemical phenotype was inherited by all of the sublines. Thus, the relative level of total cis-unsaturated fatty acids in the PG declined from 38 to 17% as a result of the transformation, and we estimated that relative levels of cis-unsaturated molecular species decreased from 76% to about 35% of the total PG (Table 1), whereas relative levels of saturated plus trans-monounsaturated molecular species increased from 24 to 65%. Changes in the composition of PG probably corresponded to those of PG in the thylakoid membranes of chloroplasts since most of the PG in leaves is found in the thylakoid membranes.

Table 1.  Fatty acid composition (mol percentage) of PG in leaves of wild-type and transgenic tobacco plants
Fatty acidsPlants
Wild-type (4)SQ7-05 (9)SQ7-11 (7)SQ8-13 (8)SQ8-15 (4)
  1. The values are means ± SD of results from repeated experiments; the number of experiments is shown in parentheses in each case. Abbreviations: 16 : 0, hexadecanoic acid (palmitic acid); 16 : 1t, 3-trans-hexadecenoic acid; 18 : 0, octadecanoic acid (stearic acid); 18 : 1, 9-octadecenoic acid (oleic acid); 18 : 2, 9,12-octadecadienoic acid (linoleic acid); 18 : 3, Δ9,12,15-octadecatrienoic acid (α-linolenic acid). aSum of the cis-unsaturated fatty acids (namely, 18 : 1 + 18 : 2 + 18 : 3). bSum of the cis-unsaturated molecular species, as calculated from Σcis-FA by a previously described method (Murata et al. 1992).

16 : 029.7 ± 4.639.5 ± 5.340.0 ± 3.437.0 ± 1.835.3 ± 0.8
16 : 1t30.4 ± 4.635.7 ± 5.635.5 ± 2.937.8 ± 1.038.2 ± 1.3
18 : 0 1.7 ± 0.4 8.3 ± 2.2 8.1 ± 0.5 8.0 ± 0.6 7.6 ± 0.4
18 : 1 5.2 ± 0.9 2.9 ± 0.6 3.1 ± 0.4 3.3 ± 0.5 3.2 ± 0.2
18 : 211.1 ± 0.8 5.2 ± 0.9 4.9 ± 0.6 5.2 ± 0.5 5.7 ± 0.3
18 : 321.8 ± 5.5 9.1 ± 1.7 8.4 ± 1.1 8.6 ± 1.010.0 ± 0.9
Σcis-FAa38.117.216.417.118.9
Σcis-MSb76.234.232.834.237.7

In an earlier analysis of the dynamics of fatty acyl chains in thylakoid membranes, we measured membrane fluidity in terms of the vsymCH2 vibration in the Fourier transform infrared (FTIR) spectrum (Szalontai et al. 2003). We showed that, below 25 °C, the lipid molecules in thylakoid membranes from tobacco plants that expressed squash GPAT were more rigid than those from wild-type plants. By contrast, above 25 °C, there were no significant differences  in  terms  of  the  fluidity  of  lipid  molecules between the two types of plant. In the present study, we examined the effects on the sensitivity of plants to low temperature of the rigidification of thylakoid membranes that resulted from transformation with GPAT cDNA.

Reduced growth at low temperatures

We investigated the effects of transformation with GPAT cDNA on growth of tobacco at low temperatures by examining the gross appearance of plants and their biomass (Fig. 2 and Table 2). When plants had been grown for 1 month at 25 °C, there were no differences in either gross morphology or biomass production between wild-type and transgenic plants. At 20 °C, by contrast, although the growth of wild-type plants was not significantly affected, the growth of SQ7-05 and SQ8-13 transgenic plants was severely repressed, and SQ7-05 plants developed chlorosis (Fig. 2). Biomass production by transgenic plants was also markedly reduced at 15 and 20 °C but not at 25 °C (Table 2). At 10 °C, seeds of both wild-type and transgenic plants germinated, and cotyledons, stems and roots developed. However, at this temperature, the latter plants became chlorotic and failed to thrive (Fig. 2). Seeds from transgenic plants germinated as rapidly as those of wild-type plants at chilling temperatures, such as 10, 15 and 20 °C (data not shown). Therefore, the difference in biomass production between wild-type and transgenic plants was caused by a difference in the sensitivity of plants to chilling during their development after germination. Our results indicated that the increase in saturated plus trans-monounsaturated molecular species of PG in chloroplasts strongly inhibited the growth of seedlings but not the differentiation of organs, when the transgenic plants were exposed to chilling temperatures.

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Figure 2. Effects of low temperature on the growth of wild-type and transgenic tobacco plants. Seeds were allowed to germinate and seedlings were allowed to grow at various temperatures, namely, 10, 15, 20 and 25 °C, under a 16-h light/8-h dark cycle. Photographs were taken after growth for 1 month.

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Table 2.  Effects of temperature on biomass production (mg seedling−1) by wild-type and transgenic tobacco plants
Temp. of treatmentFresh weightDry weight
Wild typeSQ7-05SQ8-13Wild typeSQ7-05SQ8-13
  1. After 1 month of growth at the indicated temperature, the fresh and dry weights of individual plants were determined. Values are means ± SD of results from two independent experiments (n = 30 plants in each).

15 °C10.0 ± 0.7 7.2 ± 0.5 4.9 ± 0.5 2.3 ± 0.31.5 ± 0.4 1.3 ± 0.1
20 °C208 ± 23 30 ± 3 50 ± 710.7 ± 1.14.6 ± 1.2 6.3 ± 0.0
25 °C305 ± 23293 ± 19276 ± 2211.2 ± 1.09.5 ± 0.011.0 ± 0.1

Absence of any effects of transformation on the structure of chloroplasts

We examined the effects of transformation on the architecture of chloroplasts by electron microscopy, which revealed that there were no significant differences in terms of the size and architecture of chloroplasts between wild-type and transgenic plants (Fig. 3a, c & e). Furthermore, the structure of thylakoid membranes was similar in wild-type and transgenic plants (Fig. 3b, d & f). Thus, we can infer that changes in PG might not alter the phenotypic morphology of organelles at the normal growth temperature, namely, 25 °C.

image

Figure 3. Electron micrographs of chloroplasts in leaves from wild-type and transgenic tobacco plants. (a) Wild-type tobacco; (c) SQ7-05 transgenic tobacco; (e) SQ8-13 transgenic tobacco. Panels (b), (d) and (f) show magnified views of thylakoid membranes in panels (a), (c) and (e), respectively. Bars correspond to 1.7 µm in panels (a), (c) and (e), and 0.27 µm in panels (b), (d) and (f).

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Damage to inflorescences at low temperatures

Among the various stages of the life cycle of plants, the reproductive stage is particularly sensitive to low-temperature stress (Boyer 1982; Rhoades & Loveday 1990). We examined the effects of low-temperature treatment on the gross appearance of inflorescences of wild-type and transgenic plants (Fig. 4). Before low-temperature treatment, the morphology of the inflorescences of wild-type and transgenic plants that had been grown at 25 °C was normal. When plants with inflorescences were grown at 5 °C for 1 week, most of the flower buds were abscised from wild-type plants, whereas the flowers and fruits were affected to only a limited extent. By contrast, the inflorescences of SQ7 and SQ8 plants were severely damaged; almost all of the flower buds were abscised and all the flowers died. At 10 °C, abscission of young flower buds occurred similarly in wild-type and transgenic plants. However, the buds that remained on the plants were still able to produce flowers and fruits. At 15 °C there were no obvious deleterious effects on either type of plant, despite the retardation of plant growth. These results revealed that increased levels of saturated and trans-monounsaturated molecular species of PG in chloroplasts had a significant negative effect on the cold tolerance of inflorescences.

image

Figure 4. Effects of low temperature on inflorescences of wild-type and transgenic plants. Plants that had been grown in pots under normal conditions at 25 °C for 75 d were grown at a low temperature, namely, 5, 10 or 15 °C, for 7 d and then grown under normal conditions for 2 d at 25 °C prior to photography. All treatments were performed under a 16-h light/8-h dark cycle. Bars correspond to 5 cm.

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Table 3 shows quantitatively the results of the effects of low-temperature treatment on inflorescences of wild-type and transgenic plants. When wild-type plants were exposed to 5 °C for 7 d and subsequently to 25 °C for 2 d, the mean number of flower buds decreased from 12 to four as a result of the abscission of flower buds. By contrast, the number of flowers did not change and the number of fruits increased. These results suggest that, among the three types of reproductive organ, the flower buds were the most sensitive to low temperature and that, once the fruits and flowers had formed, they were resistant to the effects of low-temperature stress. When transgenic plants were grown at 5 °C for 7 d, the numbers of flower buds and flowers decreased. These results reflected the abscission of flower buds and the death of flowers.

Table 3.  Effects of low temperature on the numbers (number plant−1) of flower buds, flowers and fruits on wild-type and transgenic plants
Temp. of treatmentWild typeSQ7-05SQ8-13
BudFlowerFruitBudFlowerFruitBudFlowerFruit
  1. Plants were grown in pots under normal conditions at 25 °C for approximately 75 d until there were three or four inflorescences on each plant. For low-temperature treatment, plants were grown at 5, 10 or 15 °C for 7 d and then incubated under normal growth conditions for 2 d at 25 °C. All treatments were made under a 16-h light/8-h dark cycle. The values are means ± SD of results with five plants in each experiment.

Before stress11.8 ± 4.57.8 ± 2.7 1.0 ± 1.113.9 ± 1.310.5 ± 4.9 1.4 ± 1.912.3 ± 2.39.9 ± 2.3 1.0 ± 1.4
 5°C 4.0 ± 2.06.3 ± 3.9 3.8 ± 1.5 1.3 ± 1.2 0.0 ± 0.0 1.0 ± 1.0 0.0 ± 0.00.0 ± 0.0 0.0 ± 0.0
10°C 4.3 ± 1.76.5 ± 3.1 7.0 ± 1.2 4.0 ± 0.0 9.5 ± 3.5 5.5 ± 0.7 0.0 ± 1.40.5 ± 3.5 5.0 ± 1.4
15°C12.5 ± 3.77.5 ± 3.411.0 ± 4.5 6.3 ± 3.4 9.8 ± 3.810.0 ± 4.2 8.0 ± 2.88.0 ± 5.712.0 ± 0.0

At 10 °C, the damage to the three types of reproductive organ was less severe than at 5 °C. However, transgenic plants were much more strongly affected by low temperature than wild-type plants. When wild-type plants were grown at 15 °C for 7 d, the number of flower buds and flowers did not change and the number of fruits increased. These results indicate that new flower buds were produced during the incubation at 15 °C and that the flower buds and flowers developed into flowers and fruits, respectively. When transgenic plants were grown at 15 °C for 7 d, the number of flower buds decreased but the number of fruits increased, as it did in wild-type plants. The number of flowers remained unchanged. Therefore, at 15 °C, only the production of flower buds was affected in transgenic plants, as compared with wild-type plants. These results clearly demonstrated that the extent of saturation and trans-monounsaturation of PG determined the sensitivity of tobacco plants to low temperature.

During this series of experiments, we also found that the inflorescence meristems were the most sensitive to low temperature of all the reproductive organs. Table 4 shows that, when transgenic plants were exposed to low temperatures, such as 5 and 10 °C, all the inflorescence meristems were abscised. At 15 °C, the abscission was much less significant than at 5 and 10 °C. The inflorescence meristems of wild-type plants were less sensitive to low temperature than those of transgenic plants; Although more than half of the meristems were abscised at 5 °C, no abscission of the inflorescence meristems was observed at 10 and 15 °C. These observations suggest that the saturation of PG is important in the sensitivity of inflorescence meristems to low temperatures.

Table 4.  Effects of low temperature on the abscission of inflorescence meristems (number of inflorescence meristems plant−1) from wild-type and transgenic plants
Temp. of treatmentWild-typeSQ7-05SQ8-13
AttachedAbscisedAttachedAbscisedAttachedAbscised
  1. Plants were grown and treated as described in Table 3. The values are means ± SD of results for five plants in each experiment.

Before stress3.0 ± 0.03.7 ± 0.53.6 ± 0.5
5 °C1.0 ± 0.82.0 ± 0.80.0 ± 0.03.7 ± 0.50.0 ± 0.03.6 ± 0.5
10 °C3.3 ± 0.20.0 ± 0.00.5 ± 0.73.2 ± 0.70.0 ± 0.03.6 ± 0.5
15 °C4.3 ± 0.50.0 ± 0.02.0 ± 1.21.7 ± 1.53.0 ± 0.00.6 ± 0.5

CONCLUSION

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

In this study, we established homozygous lines of transgenic tobacco plants, in which transgenes were located at a single locus, as indicated by the segregation of antibiotic resistance and Southern blotting analysis (Fig. 1). Thus, we were able consistently to reproduce the same results in plants, grown from seeds, over several generations at least.

An analysis of fatty acids in PG indicated that the transformation had increased the relative level of saturated plus trans-monounsaturated molecular species of PG from 24 to 65% (Table 1). In a previous study, FTIR spectrometric analysis of the physical state of thylakoid membranes from leaves revealed that this change in PG decreased membrane fluidity at physiological temperatures and raised the critical temperature for the phase transition from the liquid-crystalline to the phase-separated state (Szalontai et al. 2003).

The life cycle of plants includes many stages, from the germination of seeds to the production of seeds by mature plants. Our study showed that an increase in the relative level of saturated and trans-monounsaturated molecular species in PG increased the sensitivity of tobacco plants to low temperature during the growth of young seedlings (Fig. 2 and Table 2) and the maturation of reproductive organs (Fig. 4, Tables 3 & 4). Among the reproductive organs, the sensitivity to low temperature increased in the order: fruits < flowers < flower buds < inflorescence meristems. When compared with that of wild-type plants, the sensitivity to low temperature of transgenic plants was consistently higher in all the reproductive organs.

ACKNOWLEDGMENTS

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

We are grateful to Ms Yu Kurata and Ms Fumiko Yamashiro (National Institute for Basic Biology) for the culture of plants. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (A) to A.S. (no. 13740465) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Cooperative Research Program of the National Institute for Basic Biology (Japan) for Studies of the Molecular Mechanisms of Stress Tolerance in Plants. B.Y.M. was supported financially by a grant from the Korea Science and Engineering Foundation (KOSEF), Taejeon, Korea, and R.S. was the recipient of a postdoctoral fellowship for foreign researchers from the Japan Society for the Promotion of Science (no. P-01108).

REFERENCES

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