Journal of Experimental Botany, Vol. 55, No. 396, pp. 387-395, February 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells
Received 13 June 2003; Accepted 16 October 2003
Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0101, Japan
* To whom correspondence should be addressed. Fax: +81 743 72 5469. E-mail: kazz{at}bs.aist-nara.ac.jp
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Abstract |
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The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control. In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants. Expressions of both AtHAL3 genes are induced by salt stress. AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance. In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity. This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3. To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified. NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined. Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells. NtHAL3 genes could complement the temperature-sensitive mutation in the E. coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway. Cells overexpressing NtHAL3a had an increased intracellular ratio of proline. Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells.
Key words: HAL3 genes, HAL3 proteins, overexpression, proline biosynthesis, salt tolerance, tobacco cells.
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Introduction |
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The HAL3/SIS2 gene of Saccharomyces cerevisiae was identified by its ability to confer salt tolerance to wild-type cells in the presence of toxic concentrations of sodium chloride. Intracellular levels of sodium and potassium were dependent on the level of HAL3 expression. Expression of ENA1/PMR2A, a gene encoding the plasma membrane Na+-ATPase involved in sodium and lithium efflux, is negatively regulated by both the Hal3 and Ppz1 signal transduction pathways, and positively regulated by a calcineurin-dependent pathway. Hal3 protein directly interacts with Ppz1 protein and inhibits its protein phosphatase activity (Mendoza et al., 1994; Posas et al., 1995; Ferrando et al., 1995; Marquez and Serrano, 1996; de Nadal et al., 1998). It has also been reported that overexpression of HAL3/SIS2 suppressed the growth defect and stimulated G1 cyclin expression in a type-2A protein phosphatase sit4 mutant (Di Como et al., 1995; Sutton et al., 1991; Fernandez-Sarabia et al., 1992). These data suggest that Hal3 protein is a multifunctional regulator involved in salt tolerance, cell cycle control and cell wall integrity via its interaction with the regulatory subunit of Ppz1 protein (de Nadal et al., 1998; Clotet et al., 1999). It has also been suggested that these regulatory activities are dependent on intracellular K+ concentration and pH which are mediated by a potassium transport system involving the Trk1 and Trk2 proteins (Yenush et al., 2002).
In plants, two HAL3 homologue genes were isolated from Arabidopsis thaliana and characterized. The expression of these genes is increased under conditions of salt stress. Transgenic Arabidopsis overexpressing the AtHAL3a gene showed improved tolerance to salt and osmotic stress (Espinosa-Ruiz et al., 1999). Although these data suggest some relationships between the AtHAL3a gene and osmolyte accumulation and/or transport of toxic sodium ions, the molecular function of plant HAL3 protein in salt tolerance is still unknown. Recently, it was reported that AtHAL3a protein containing a flavin mononucleotide catalysed the reaction from the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in vitro (Espinosa-Ruiz et al., 1999; Kupke et al., 2001). This reaction is known to be involved in the coenzyme A biosynthesis pathway of E. coli (Kupke et al., 2000; Begley et al., 2001). Kupke et al. (2001) suggest that the AtHAL3a protein functions in the coenzyme A biosynthetic pathway of plants, and is not involved in signal transduction like the yeast Hal3 protein. To investigate this hypothesis, a cultured tobacco cell system was chosen to examine the metabolic change in cells caused by the overexpression of plant HAL3. Nicotiana tabacum L.cv. Bright Yellow-2 (BY2) cells are fast growing, highly homogenous (Nagata et al., 1992) and can be easily transformed. Since it is considered that the homogenous cell culture system is suitable for the analysis of metabolic change in plant cells, BY2 cells were chosen to investigate the change in amino acid levels caused by the overexpression of HAL3 gene(s).
In this report, three HAL3 homologues were isolated from N. tabacum. The NtHAL3 genes were constitutively expressed in all organs examined, regardless of the conditions of stress in the tobacco plant. Salt, hyperosmotic, and lithium tolerance were improved by overexpression of NtHAL3a in BY2 cells. This indicates that the NtHAL3a gene is functionally homologous to AtHAL3a. It is also reported that the NtHAL3 gene could complement the temperature-sensitive dfp mutation of E. coli, and proline levels were increased in NtHAL3a overexpressing BY2 cells.
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Materials and methods |
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Plant materials, culture and transformation
N. tabacum L. cv. SR-1 was grown in soil in a temperature-controlled greenhouse at 25 °C with a 16/8h light/dark cycle. Culture conditions and transformation of cultured tobacco BY2 cells has been described previously (Nakayama et al., 2000).
Isolation of NtHAL3 cDNAs
Total RNA was extracted from exponentially growing BY2 cells and from the shoot apex of tobacco SR-1. Poly (A) RNA was purified by the Oligotex-dT30 kit (Takara Bio Ink, Otsu, Japan), and cDNA libraries were constructed in the ZIP-LOX vector (GibcoBRL, Rockville, MD, USA). Plaques were replicated on nylon membranes (Hybond-N+, Amersham Pharmacia, Piscataway, NJ, USA), and hybridization was performed at 65 °C in buffer as described previously (Church and Gilbert, 1984). The BY2 cDNA library was screened with a 32P-labelled partial fragment of AtHAL3 cDNA (40–594 bp, accession no. U80192
[GenBank]
, 1997), and five positive clones were isolated from the BY2 cDNA library. The SR-1 cDNA library was subsequently screened with 32P-labelled NtHAL3a cDNA that was isolated from BY2, and 11 positive clones were isolated.
Detection of mRNA expression of the NtHAL3 genes
Total RNA was extracted from each sample (various organs of 4–6-week-old tobacco) as previously described for Northern hybridization (Nakayama et al., 2000) or by the AGPC method for RT-PCR (Chomczynski et al., 1987). For RT-PCR analysis, first-strand cDNA was synthesized from 1 µg of total RNA with a first-strand cDNA kit (Perkin Elmer, Branchburg, NJ, USA), and was subsequently used as the template for the PCR reaction. PCR was performed with the following cycle parameters using specific primer sets; once at 94 °C for 2 min; 29 and 31 cycles for NtHAL3a and NtHAL3c or 32 and 34 cycles for NtHAL3b at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; and once at 72 °C for 5 min. The specific primer sets are NtHAL3a (5'-CAGAGATGGAACCGGTTCAG ATT-3' and 5'-GCGTCATAATAGAGTCTTACAGCTTGGA-3'), NtHAL3b (5'-GGTGCAGTAAAGAATCCTTTTCGATG-3' and 5'-CATGACCTGTGGATCACGA-3'), or NtHAL3c (5'-GTCCTGTT CGTTCCGTCGA-3' and 5'-GTGCGTCATAATAGATTCTTACA GCTTGAT-3'). Actin was used as an internal control, and the set was 5'-CCTCTTAACCCGAAGGCTAA-3'and 5'-GAAGGTTGG AAAAGGACTTC-3'. PCR reaction was performed using the following parameters; once at 94 °C for 2 min; 28 and 30 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; and once at 72 °C for 5 min.
Complementation test with a mutant strain of E. coli
For the complementation test with an E. coli mutant, the NtHAL3 coding sequences flanked by BamHI and XhoI sites were cloned using the BglII and XhoI cloning sites of pKC7 (Rao and Rogers, 1979). NtHAL3 mutated sequences flanked by EcoRI and SacI sites were cloned using the EcoRI and SacI cloning sites of each pZL1::NtHAL3cds plasmids, and subsequently these plasmids were used as the next PCR template to amplify the mutated NtHAL3 sequences flanked by EcoRI and XhoI sites. The NtHAL3 mutated sequences were cloned using the EcoRI and XhoI cloning sites of pKC7. Primer set for NtHAL3 coding sequences; NtHAL3a (a-BamHI-F, 5'-CGGGATCCATGGAGACTTCAGAGATGG-3' and a,c-XhoI-R, 5'-AATCTCGAGTCACGCCACGTTGCTG-3'), NtHAL3b (b,c- BamHI-F, 5'-CGGGATCCATGGAGCCTATGA CTTCAGAG-3'and b-XhoI-R, 5'-CCGCTCGAGTCACGACACG TTGCTGCC-3') or NtHAL3c (b,c-BamHI-F and a,c-XhoI-R). Mutagenesis primer sets for active-site mutation; NtHAL3a (a-EcoRI-F, 5'-CCGGAATTCATGGAGACTTCAGAGATGG-3' and muHN-SacI-R, 5'-CTCCGGAGCTCGATGTTTAG-3'), NtHAL3b (b,c-EcoRI-F, 5'-GGAATTCATGGAGCCTATGACTT and muHN-SacI-R), NtHAL3c (b,c-EcoRI-F and muHN-SacI-R). The constructs were introduced into the E. coli temperature-sensitive dfp mutant strain BW369 (Spitzer and Weiss, 1985). Transformants were cultured on solid LB Amp plates at 30 °C for 24 h or 42 °C for 12 h to examine complementation of the dfp mutation.
Construction of binary plasmids
Full-length NtHAL3a cDNA in pZL1 was digested with SalI and NotI, and subsequently cloned into the XhoI and NotI cloning sites of pMS1 which is removed by the hygromycin resistance gene from pMSH1 (Kawasaki et al., 1999).
Analysis of stress tolerance
6-day-old BY2 cells were harvested in a 50 ml centrifuge tube and centrifuged for 5 min at 800 g. After removal of the culture medium, the cell density was adjusted to 50% (v/v) with fresh medium. Five millilitres of cells were transferred to a 300 ml Erlenmeyer flask containing 95 ml of modified LS medium (CaCl2.2H2O, 0.3 mM) containing 100 µg ml–1 kanamycin and 250 µg ml–1 carbenicillin with 100 or 140 mM NaCl for salt stress, 30 or 60 mM LiCl for Li+ stress, 150 or 300 mM sorbitol for osmotic stress for 5 d.
Measurement of amino acid content in BY2
Total free amino acids were extracted from 1 g fresh weight of cells grown in a no-stress medium for 5 d. Cells were homogenized in liquid nitrogen in 5 ml of a methanol:chloroform:water mixture (12:5:2, by vol.) following previously described methods (Nakayama et al., 2000). After centrifugation at 2300 g for 5 min, the supernatant was collected in a 50 ml centrifuge tube and the extraction procedure was repeated twice. 10 ml of chloroform and 5 ml of water were added to the pooled extracts and mixed vigorously before being centrifuged at 2300 g for 5 min. The aqueous layer was collected in a 50 ml centrifuge tube and the organic layer was re-extracted with an additional 5 ml of water. The pooled aqueous layer was evaporated at 80 °C for 2 d and then dissolved in 0.8 ml of water and filtered through centrifugal filter units (0.2 µm pore; Ultrafree-MC, Nihon Milipore Ltd., Tokyo, Japan). Amino acid analysis was performed with an amino acid analyser (Model L-8500, Hitachi Ltd., Tokyo, Japan) with ninhydrin reaction of samples.
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Isolation of NtHAL3 cDNAs
To isolate tobacco HAL3 homologues, a tobacco BY2 cDNA library was screened with a partial AtHAL3a cDNA fragment as a probe. Out of approximately 400 000 plaques, five positive cDNA clones were isolated. The sequence of the longest cDNA showed homology with that of the AtHAL3a cDNA used as probe; this cDNA was named NtHAL3a. The NtHAL3a gene encodes a protein with 207 amino acid residues with a predicted molecular mass of 22.8 kDa and theoretical pI of 4.99 (Fig. 1a). Southern blot analysis suggested the presence of additional HAL3 homologues in BY2 cells and the tobacco plant SR-1 (Fig. 2). Therefore, the BY2 cDNA library was re-screened, but no other HAL3 homologues were obtained. In order to characterize these putative HAL3 homologues further, a cDNA library derived from SR-1 shoot apex cells was screened with the cDNA fragment of NtHAL3a coding region as a probe. Eleven positive clones were isolated from approximately 200 000 plaques. Three of these clones were identical in sequences to that of the previously isolated NtHAL3a cDNA. The additional cDNA clones contained two different HAL3 homologues; these genes were named NtHAL3b and NtHAL3c, respectively. Both genes encode proteins of 208 amino acids with predicted molecular masses of 23 kDa, but the theoretical pI of NtHAL3b and NtHAL3c proteins were 5.51 and 5.61, respectively (Fig. 1a). NtHAL3 proteins contain the conserved domain present in the middle region of yeast Hal3 protein (Leu-353 to Asn-435), but the aspartate- and glutamate-rich region in the carboxyl terminus of yeast Hal3 is not present in NtHAL3 (Fig. 1a). The predicted amino acid sequences of tobacco and Arabidopsis HAL3 proteins demonstrate a high degree of homology (Fig. 1b).
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Expression of the NtHAL3 genes
To investigate the mode of NtHAL3 expression, northern blot analysis was performed using the coding region of NtHAL3a cDNA as a probe. The presence of NtHAL3 mRNAs was observed in all organs of the tobacco plant, including roots, stem, young leaves, the shoot apex, and flowers (Fig. 3a). The level of NtHAL3 mRNA did not change from 1–24 h after 100 mM NaCl treatment (Fig. 3b). Northern analysis detected the total mRNA derived from all three NtHAL3 genes because the coding sequence of NtHAL3a was used as a probe. The coding sequence of the NtHAL3 cDNAs are more than 96% identical, but are less than 29% identical in their 5' untranslated regions. Although northern analysis was carried out with a DNA probe corresponding to the 5' untranslated sequence of each gene, the mRNA signal of each NtHAL3 was not clearly identified. In order to identify the expression patterns for each NtHAL3 gene under stress conditions, RT-PCR analysis was performed with primer pairs specific for the DNA sequence corresponding to the 5' untranslated regions and the coding region of NtHAL3b and NtHAL3c, and to the coding region of the NtHAL3a. The amount of detected NtHAL3 isoform mRNA did not change after treatment of cells with 100 mM NaCl or a shortage of potassium (Fig. 3c).
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Overexpression of NtHAL3a improved salt, LiCl, and sorbitol stress tolerance in BY2 cells
To investigate the molecular function of plant HAL3 genes using the transgenic BY2 system, NtHAL3a was placed under the control of the CaMV 35S promoter and introduced into BY2 cells by A. tumefaciens-mediated transformation. Approximately 80 kanamycin-resistant calli were isolated, and some calli were checked for integration and expression of NtHAL3a by genomic PCR and northern blot analysis, respectively (data not shown). Expression of NtHAL3a varied widely, and three clones were selected for further analysis (SA3, SA4, and SA5; Fig. 4a). Growth inhibition of transgenic BY2 clones by salt stress, at 100 or 140 mM NaCl was observed. Although the growth of control transformed cells (EGFP) was markedly inhibited by NaCl stress, overexpression of NtHAL3a reduced the growth inhibition in direct relation to the level of NtHAL3a expression in transgenic BY2 cells (Fig. 4b). High salt concentrations can affect cell growth negatively through hyperostmotic stress and/or direct toxicity of the sodium ion. In order to identify which aspect of salt stress NtHAL3 overexpression was affecting, growth inhibition experiments were carried out under hyperosmotic conditions with sorbitol and sodium-ion stress conditions with LiCl as a more toxic analogue of sodium (Serrano, 1996). The reduction of growth inhibition by LiCl and sorbitol stress was observed in NtHAL3a expressing BY2 cells (Fig. 4b). These results indicate that NtHAL3a relates to both hyper-osmotic stress and sodium ion toxicity in tobacco cells.
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NtHAL3 genes complement the E. coli dfp mutation
The predicted amino acid sequences of the three NtHAL3 proteins contain four highly conserved motifs (I–IV; Fig. 1a) that form a domain of 4'-phosphopantothenoylcysteine decarboxylase (PPC-DC). These motifs are also found in AtHAL3a protein, and PPC-DC activity of AtHAL3a was shown biochemically by an in vitro study (Kupke et al., 2001). PPC-DC is encoded by the dfp gene in E. coli and plays an important role in the coenzyme A biosynthetic pathway (Fig. 5a; Kupke et al., 2000; Kupke 2001; Strauss et al., 2001). To investigate whether NtHAL3 protein has PPC-DC activity, the ability of NtHAL3 to complement a temperature-sensitive dfp mutant of E. coli, a lethal phenotype at 42 °C (Spitzer et al., 1985, 1988), was examined. The three NtHAL3 genes were ligated into the E. coli expression vector pKC7 and introduced into the dfp mutant. Each NtHAL3 gene could complement the dfp mutant strain at 42 °C (Fig. 5b). Previous studies carried out in vitro demonstrated that an AtHAL3 mutant protein with a His-90 to Asn change in the PPC-DC active site lost all PPC-DC activity (Kupke et al., 2001). Therefore, the PPC-DC activity of active site mutants of NtHAL3, i.e. that NtHAL3a-H88N, NtHAL3b-H89N, and NtHAL3c-H89N are substituting the corresponding His residue by Asn, was examined by the complementation test with the E. coli dfp mutant. The three mutant NtHAL3 genes did not complement the dfp mutation of E. coli (Fig. 5c). The functional difference between the yeast Hal3 and plant HAL3 was discussed (Kupke et al., 2001). It was examined whether the yeast Hal3 protein had PPC-DC activity by a complementation test with the E. coli dfp mutant. As a result, yeast HAL3 was not able to complement the dfp mutant of E. coli (Fig. 5c). These results strongly suggest that plant HAL3 proteins have PPC-DC activity, but yeast HAL3 protein, a known regulator of protein phosphatase activity, does not possess PPC-DC activity.
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dfp is a negative control with an empty vector. Transformants were grown on LB Amp plates at 30 °C for 24 h or 42 °C for 12 h.Overproduction of NtHAL3a increases the intracellular ratio of proline
Coenzyme A and its thioesters are essential cofactors for many enzymatic and energy-yielding reactions including the TCA cycle, fatty acid metabolism, and amino acid metabolism (Abiko, 1975; Tahiliani and Beinlich, 1991; Begley et al., 2001). If the PPC-DC activity of the NtHAL3 protein functions in the metabolic pathway of coenzyme A biosynthesis from pantothenate, the intracellular concentration of some of the downstream metabolites of this pathway may possibly be increased in NtHAL3a overexpressing cells. Furthermore, hyperosmotic stress tolerance was improved in the transgenic BY2 cells. These results suggest the possible accumulation of amino acids that could function as a compatible solute, i.e. proline and citrulline (Delauney and Verma, 1993; Yoshiba et al., 1997; Akashi et al., 2001). Therefore, an attempt was made to determine the intracellullar free amino acids concentration in NtHAL3a overexpressing BY2 cells. Table 1 shows the free amino acid contents in clone SA3 of transgenic BY2 under no-stress conditions. The percentage of proline in SA3 cells increased 4.4-fold compared with that in control cells under non-stress condition (Table 1), and this increased level of proline was maintained at approximately three times higher than control even under salt-stress conditions (SA3, 4.1%; EGFP, 1.4%). The percentage of proline in another clone, SA4, was 4.9 times higher than that in control cells (data not shown).
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Discussion |
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Three tobacco HAL3 homologue genes, NtHAL3a, NtHAL3b, and NtHAL3c, were isolated with using Arabidpsis AtHAL3 cDNA as a probe. The NtHAL3a gene was cloned from cultured BY2 cells, and the others were cloned from the SR-1 tobacco plant. Although BY2 and SR-1 were different cultivars, a partial cDNA fragment of NtHAL3a was isolated from a cDNA library of SR-1 (data not shown). Results of Southern blot analysis suggested the possibility that there are additional HAL3 homologue genes in the tobacco plant SR-1. The expression of the HAL3 gene was regulated by salt concentration in Arabidopsis, but the NtHAL3 genes were constitutively expressed in all organs, regardless of salt-stress conditions (Fig. 3b, c) in the tobacco plant. This difference is possibly important when considering the function of HAL3 genes in the salt tolerance of plant cells.
NtHAL3a overexpression negated the growth inhibition of BY2 cells by lithium and sodium stress (Fig. 4b, c). Lithium and sodium ion contents were examined in the transgenic cells, but these intracellular ion contents were not very different between control cells and NtHAL3a overexpressing cells under LiCl or NaCl stress conditions (data not shown). Therefore, overexpression of NtHAL3a probably does not affect ion homeostasis regulation in plant cells. It is known that sodium and lithium ion stress cause superoxide anion and free radical formation in BY2 cells (Kawano et al., 2001; Hong et al., 2000). NtHAL3a overexpressing cells showed approximately a 4–5-fold increase in the intracellular ratio of proline compared with the control cells with and without salt-stress conditions (Table 1). Previous reports proposed that proline can function as a free radical scavenger under salt and heavy metal stress conditions (Smirnoff and Cumbes, 1989; Kishor et al., 1995; Nanjo et al., 1999; Hong et al., 2000; Siripornadulsil et al., 2002). The increased level of proline caused by NtHAL3 overexpression could possibly lead to improved salt-tolerance of transgenic BY2 cells by acting as a free radical scavenger. The scavenging ability of free radicals in NtHAL3a expressing cells should be compared with that of control cells in future experiments.
The NtHAL3 proteins contain four conserved motifs from PPC-DC, an enzyme that functions in the coenzyme A biosynthetic pathway, and NtHAL3 genes can complement an E. coli temperature-sensitive dfp mutation (Fig. 5b). Furthermore, active site mutant genes of NtHAL3 could not complement the dfp mutation (Fig. 5c). These results proved the hypothesis, based on the results of in vitro experiments by Kupke et al. (2001), that the plant HAL3 protein functions in the coenzyme A biosynthetic pathway, by an in vivo experiment. Acetyl-CoA is involved in amino acid synthesis at several points including providing the carbon skeletons for leucine, a series of acetylated intermediates for the synthesis of ornithine and arginine, or through the TCA cycle (Ireland, 1997; Thompson, 1980; Yokota et al., 2002). Therefore the intracellullar free amino acid content of NtHAL3a transgenic BY2 cells was determined. The percentage of proline was increased about 4–5-fold in NtHAL3a transgenic cells with or without stress (Table.1). Proline is synthesized from both glutamate and ornithine in plant cells (Delauney and Verma, 1993), and pathway selection depends on the developmental stage of the plant and environmental stress (Delauney et al., 1993; Roosens et al., 1998). Acetyl-CoA is used as the first metabolic substrate in the ornithine pathway (Ireland, 1997; Thompson, 1980; Yokota et al., 2002). In the NtHAL3a overexpressing cells, the percentage of proline was increased and that of arginine, also synthesized by the ornithine pathway, was increased approximately 2-fold (Table 1). Therefore, it is thought that the ornithine biosynthesis pathway may be strengthened by an increase in PPC-DC activity, and, consequently, the proline and arginine ratio was increased in NtHAL3a overexpressing cells. It will be important to determine whether the production level of coenzyme A and/or acetyl-CoA is increased by overexpression of NtHAL3a in BY2 cells. The intracellular localization of NtHAL3 proteins will be important information for understanding the proper use of each NtHAL3 in tobacco cells. Based on the present study, it appears that plant HAL3 genes are not only important for the molecular breeding of salt-tolerant plants, but also for the metabolic regulation of coenzyme A biosynthesis from pantothenate and its effects on the biosynthesis of several amino acids through the ornithine pathway in plant cells.
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Acknowledgements |
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We are grateful to Dr Thomas Kupke, Universitat Tubingen for helpful discussions about PPC-DC. We thank Dr Bernard Weiss, Emory University for generously providing the E. coli strain BW369 and the pKC7 plasmid. We also thank Dr Masami Sekine and Dr Ko Kato in our laboratory for useful discussions. This work was supported by a grant from the Research for the Future Program (JSPS-RFTF00L01604) from the Japan Society for the Promotion of Science and a grant fom the Development of Micro HPLC for Post-genomic Analysis from the Ministry of Economy Trade and Industry, through the Kansai Bureau of Economy, Trade and Industry, and the Osaka Science and Technology Center.
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