Specific expression of glucose-6-phosphate dehydrogenase
(G6PDH) gene by salt stress in wheat (
Triticum aesti
6
um
L.)
Yasue Nemoto, Tetsuo Sasakuma*
Kihara Institute for Biological Research and Graduate School of Integrated Science,Yokohama City Uni6ersity,Maioka641-12,Totsuka-ku,
Yokohama244-0813,Japan
Received 25 October 1999; received in revised form 30 May 2000; accepted 30 May 2000
Abstract
We isolated three kinds of full-length cDNA clones for glucose-6-phosphate dehydrogenase (G6PDH) from wheat. They showed over 80% sequence homology with potato and alfalfa at the amino acid level, suggesting that the genes are highly conserved in angiosperms. The lack of a plastidic signal sequence, as well as a higher transcript accumulation level in roots than leaves, suggested that the wheat cDNA sequences encode the cytosolic isoform of the enzyme. Genomic Southern analysis revealed that the isoform is encoded by a few copies of the gene in the wheat genome. Southern analysis of nullisomic-tetrasomic lines suggested that at least one gene is located on chromosome 2B. The G6PDH transcript started to accumulate in the roots after 2 h of 0.15 M NaCl treatment, reaching its maximal level after 12 h of exposure. The G6PDH gene did not respond to either mannitol or abscisic acid (ABA) treatment. The NaCl-specific early response of the G6PDH gene is discussed in relation to the putative role of the enzyme in the salt stress response in plants. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:ABA; cDNA; Glucose-6-phosphate dehydrogenase; Osmotic stress; Salt stress;Triticum aesti6umL.
www.elsevier.com/locate/plantsci
1. Introduction
Salt stress induces various biochemical and physiological responses in plants [1 – 4]. For exam-ple, the level of abscisic acid (ABA) increases as a result of salt stress. Exogenous application of ABA accelerated the adaptation of cultured cells to salt [5] and cold stress [6], providing further support for a role of ABA in the adaptation of plants to osmotic stress. Salinity stress includes not only ionic toxicity but also osmotic alteration [7]. In order to understand the plant responses to salt stress, it is necessary to identify the genes involved in the salt response and to verify their individual contributions to the response. We previ-ously isolated five wheat early salt-responding (WESR) genes, that became activated after
expo-sure to 0.15 M NaCl for 2 h. We found that one of the cDNA fragments, WESR5, shows sequence homology to glucose-6-phosphate dehydrogenase (G6PDH) gene [8].
G6PDH (EC 1.1.1.49) is a key enzyme that catalyzes a non-equilibrium reaction, and thus reg-ulates the flux of carbon through the pentose-phosphate pathway. It is also a key enzyme in that it catalyzes the first step of the oxidative pentose-phosphate pathway. The main function of the pentose-phosphate pathway is to provide NADPH and other intermediates, such as pentose and ery-throse-4-phosphate [9]. It was reported that at least two isoforms of G6PDH exist in green plant tissues, one in the cytosol and the other in chloro-plasts [10]. cDNA clones for plant G6PDH have been isolated from two species, potato [11] and alfalfa [12], but not yet from monocots.
Recently, G6PDH was reported to be involved in some metal-induced and other stress responses in plants, as the specific activity of G6PDH was * Corresponding author. Tel.: +81-45-8201902; fax: +
81-45-8201901.
E-mail address:[email protected] (T. Sasakuma).
found to be modulated by metals such as zinc, cadmium [13], and aluminium [14]. In the case of aluminium stress, a rapid increase in G6PDH activity was observed in Al-resistant wheat culti-var [14]. Also exposure of alfalfa cells in suspen-sion to an elicitor from yeast cell walls resulted in an increased transcription rate for the G6PDH gene [12].
In this study, we report the isolation of full-length cDNA clones for G6PDH from wheat. We demonstrated that transcriptional activation of the gene specifically occurred response to NaCl stress, but not to either osmotic stress or exogenous ABA treatment.
2. Materials and methods
2.1. Plant material and stress treatments
Wheat plants (Triticum aesti6um L. cv. Chi-nese Spring) were grown in a growth chamber under conditions described previously [8]. Two-week-old seedlings were transferred to fresh Linsmaier-Skoog medium [15] supplemented with 0.15 M NaCl or 5.0% mannitol. The osmotic potential of 5.0% mannitol was evaluated to be equivalent to that of 0.15 M NaCl by measure-ment with an osmometer (OM-801, Vogel, Ger-many). For ABA treatment, 2-week-old seedlings were transplanted in the above medium contain-ing 20 mM ABA dissolved in dimethylsulfoxide (DMSO). To identify the location of the G6PDH gene in the wheat genome, we used nullisomic-tetrasomic stocks for homoeologous chromosomes [16].
2.2. cDNA cloning and sequence analysis
Total RNA was isolated from roots treated with 0.15 M NaCl for 6 h as described before [8]. Poly(A)+RNA was purified from the total
RNA by using Oligotex-dT30 (Takara Shuzo,
Shiga, Japan). cDNA was synthesized from the poly(A)+RNA by using cDNA synthesis kit
(Pharmacia, Uppsala, Sweden), ligated with the l Zap II vector, packaged in vitro with Giga-pack III in vitro Giga-package extract (Stratagene, La Jolla, CA), and plated on Escherichia coli strain XL1-Blue. The WESR5 clone obtained by differ-ential display [8] was radioactively labelled and
used to screen the cDNA library. Selected plaques were converted to pBluescript plasmids according to the manufacturer’s protocol (Strata-gene), and sequenced by using a DNA sequencer (model 4000; Li-Cor, Lincoln, NE, USA).
2.3. Rapid amplification of cDNA ends
2.3.1. 5%RACE
The 5% end of G6PDH cDNA sequences were
amplified by 5%RACE [17,18]. For the
gene-spe-cific primers, oligonucleotides corresponding to the 5% end region of the cDNA clone obtained
by cDNA library screening (WR5-1, 5% -AGA-GATACAGGCTTTTCCATTGC-3%) were
syn-thesized. The first-strand cDNA was synthesized by Superscript™ II RNase H− reverse
transcrip-tase (Gibco-BRL) and WR5-1 primer at 42°C for 1 h from poly(A)+RNA of roots treated
with 0.15 M NaCl for 12 h, and then incubated with RNase H at 37°C for 20 min. Then d(A)n tails were added for 10 min at 37°C using termi-nal deoxynucleotidyl transferase (Takara Shuzo) in the presence of dATP. The second strand was synthesized with RACE-N (5%
-AAGGCTC- CGTCGGCATCGATCGCGCGACTCTTTTTT-TTTTTTTTTTTT(A,G,C)-3%) primer and Ex Taq
polymerase (Takara Shuzo). PCR was carried out with the primer RACE-0 (5%
-AAGGCTC-CGTCGGCATCG-3%) and WR5-1 primers. An
aliquot of the PCR products was then subjected to another nested PCR using RACE-I (5%
-GCATCGATCGCGCGACTC-3%) and WR5-1.
2.3.2. 3%RACE
The full-length cDNA of G6PDH was ob-tained by 3%RACE. Based on the sequences of
5%RACE products, gene specific 5% end primers (WR5-4, 5%
-GGGCAAGGAAGGGGCCACCC-CCCTC-3% and WR5-5, 5% -GCCACCCCCCTCC-CCATTCCTACC-3%) were synthesized. The
first-strand cDNA was synthesized from root total RNA with RACE-C (5%-CGGCATCGATC GCGCGACTCTTTTTTTTTTTTTTTTTTTT-3%)
primer. The PCR was carried out with the primer RACE-2 (5%
2.4. Genomic southern blot analysis
Total DNA was extracted from wheat leaves by the cetyltrimethylammonium bromide (CTAB) method [19]. Twenty micrograms of genomic DNA was digested with restriction enzymes, sepa-rated on a 0.85% agarose gel, denatured, and blotted onto a nylon membrane (Hybond N+,
Amersham). The full-length G6PDH cDNA (Tagpd1) was labelled with [a-32P]dCTP by
ran-dom priming using a Bca BEST™ Labeling kit (Takara Shuzo) and used as a probe. Hybridiza-tion was performed at 68°C overnight with hy-bridization buffer containing 6×SSC, 5×Denhardt’s solution, 0.5% SDS, and 10 mg/ml denatured salmon sperm DNA [20]. After hy-bridization, the membrane was washed at 68°C with 2×SSC/0.1% (w/v) SDS for 20 min and 0.2×SSC/0.1% (w/v) SDS for 20 min to control stringency.
2.5. Northern hybridization analysis
Total RNAs were isolated from roots exposed to 0.15 M NaCl, 5.0% mannitol and 20 mM ABA, and non-treated plants. These RNAs were sub-jected to northern analysis as described previously [8]. The full-length G6PDH cDNA (Tagpd1) was used as a probe. The loading amount of total RNA was standardized by comparison with the hybridization signal of wheat rRNA gene.
3. Results
3.1. Isolation of full-length G6PDH cDNAs
This study was aimed at the isolation of full-length cDNA clones encoding G6PDH by use of WESR5 [8] as a probe. A cDNA library was prepared from poly(A)+RNA from roots exposed
to 0.15 M NaCl for 6 h. Five clones out of 1.41×105 plaques were isolated and sequenced.
All of them lacked 5%regions of the sequences. To obtain the missing parts of the cDNA sequences, we employed the 5%RACE approach. Three 5%RACE sequences were obtained, and specific
primers were designed at their 5% ends. To obtain the full-length G6PDH cDNA, we performed 3%RACE using the specific primers. Finally we
obtained three kinds of cDNA clones, Tagpd1,
Tagpd2 and Tagpd3, distinguishable each other by
EcoRI and/or EcoRV digestion patterns. The nu-cleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB029454 (Tagpd1), AB029455 (Tagpd2), and AB029456 (Tagpd3). They showed 98% homology to each other at the deduced amino acid sequence level (Fig. 1). The wheat G6PDH cDNA Tagpd1, Tagpd2, and Tagpd3 encoded a polypeptide with a deduced Mr of 57.7, 58.3, and 57.7 kD, and a pI of 5.88, 5.88, and 6.43, respectively.
The deduced amino acid sequences of G6PDH from wheat were aligned with those of other or-ganisms (Fig. 1). The substrate-binding site was highly conserved among all sequences. The de-duced amino acid sequences of wheat G6PDH exhibited 81.5% identity to those of the potato cytosolic G6PDH, 75.9% to the alfalfa G6PDH, and 30 – 50% identity to G6PDHs from E. coliand yeast.
The wheat G6PDH cDNA clones, Tagpd1, Tagpd2 and Tagpd3, did not contain the plastidic signal sequence, suggesting that they encoded the cytosolic form of the enzyme.
3.2. Southern blot analysis
Genomic Southern blot of common wheat cv. ‘Chinese Spring’ (CS) was hybridized with Tagpd1 probe (Fig. 2). We detected three to seven frag-ments, suggesting that this isoform is encoded by a low number of copies of the gene in the wheat genome (Fig. 2A). In barley (Hordeum 6ulgare), G6PDH has been mapped on chromosome 2H [21], implying that the gene is also located in chromosome 2 in wheat, because of the chromo-some synteny between barley and wheat. Genomic Southern blot analysis of nullisomic-tetrasomic stocks for chromosome 2 [16] was conducted by using Tagpd1 as a probe. The fragment of 5.8 kb was not detected in nulli2B-tetra2A line, suggest-ing that at least one G6PDH gene representsuggest-ing the 5.8 kb fragment is located on chromosome 2B in wheat genome.
3.3. Temporal accumulation pattern of G6PDH mRNA in response to NaCl treatment
treatment with 0.15 M NaCl. The full-length G6PDH cDNA (Tagpd1) was used as a probe, and the transcript level of G6PDH during the time course was quantified by northern hy-bridization. The transcript accumulation of G6PDH in the roots increased after 2 h of NaCl treatment, reached maximum at 12 h, and
subse-quently declined to below the baseline level after 48 h of treatment (Fig. 3). At 12 h, the profile showed a 2.2-fold increase in the steady-state mRNA levels relative to 0 h (non)-stressed controls. The steady-state mRNA accumulation occurred at lower levels in leaves (data not shown).
Fig. 2. Genomic Southern blot analysis of G6PDH gene. Genomic DNA of ‘Chinese Spring’ (A) and nullisomic-tetra-somic lines for chromosome 2 (B) was analyzed. Genomic DNA was digested with restriction enzymes, electrophoresed on 0.85% agarose gel, blotted onto a nylon membrane, and probed with Tagpd1 cDNA. The sizes of DNA molecular mass standards are indicated on the left. The arrowhead indicates the fragment lost in the nulli2B-tetra2A (N2BT2A) genomic DNA.
4. Discussion
We previously isolated five cDNA fragments for WESR genes by the differential display method, and WESR5 clone showed sequence homology to the G6PDH gene from potato and alfalfa [8]. In this study, we obtained full-length cDNA se-quences encoding G6PDH in wheat. In plants, there are at least two isoforms of G6PDH in the cytosol and the plastidic stroma, and both iso-forms catalyze the rate-limiting step of the oxida-tive pentose-phosphate pathway. Comparison of the deduced amino acid sequences of cytosolic and plastidic isoforms of potato revealed 65% similar-ity between them [22]. The plastidic isoform has a significantly longer 5% region encoding a transit
peptide. The transcript of cytosolic G6PDH was less accumulated in the leaves than in roots [22]. The wheat G6PDH cDNAs lacked the transit peptide, and their transcripts were shown to accu-mulate at a low level in the leaves by northern analysis (data not shown). These characteristics of the wheat cDNA clones, Tagpd1, Tagpd2 and Tagpd3, suggested that the clones encoded the cytosolic isoform of G6PDH. The wheat G6PDH sequences were over 80% homologous to the se-quence of the cytosolic isoform from potato at the amino acid level [23]. The wheat cDNAs isolated in the present study are the first G6PDH genes from monocots that show over 80% sequence ho-mology to those of potato and alfalfa, suggesting that the sequences of the genes of their enzyme are highly conserved among the angiosperms.
Wheat Tagpd1 was used to determine the gene copy number and chromosomal location by Southern blot analysis. G6PDH was suggested to be encoded by a few copies of the gene in the wheat genome. As common wheat is a hexaploid with three genomes, the multiple bands observed on genomic blots could be the result of homoe-ologous variation. We determined that one of fragments digested by HindIII was located on chromosome 2B.
G6PDH transcripts showed rapid induction within 2 h of NaCl treatment and reached a peak of expression by 12 h. During the NaCl stress, the expression returned to below the baseline level, indicating that the G6PDH is involved in the initial responses of salt-stressed plants. Similar salt-response was observed for Esi clones isolated from salt-stressed wheat grass (Lophopyrum elon -3.4. G6PDH mRNA does not accumulate in
response to mannitol or exogenous ABA treatment
gatum) [24]. Esi clones were observed to be in-duced within 2 h after exposure to 0.25 M NaCl and peaked after 6 h in wheat [11,24].
It is well known that exposure to NaCl solution causes osmotic stress to plant cells. A variety of environmental stresses, such as high salt concen-tration, water deficiency and cold, trigger the syn-thesis of ABA, which in turn induces the expression of various plant genes [1,25,26]. We examined whether G6PDH genes are induced by osmotic stress and by exogenous ABA treatments, and found that the degree of the response to mannitol or exogenous ABA was negligible in comparison with that to NaCl treatment, indicat-ing that the ionic form of salt affects the gene expression of G6PDH. A similar NaCl-specific stimulation was reported for transcripts of the 70 kDa (catalytic) subunit of tonoplast H+-ATPase, which was induced by NaCl stress, but not by drought stress or ABA treatment [27]. This sug-gests that the response observed with NaCl is associated with the ionic aspect rather than with the osmotic component of NaCl stress.
A unique cis-element, called ABRE, has been characterized in the ABA-regulated genes [25]. However, regulatory mechanism of salt-induced gene expression has not yet been clarified in plants. Changes in turgor, cytosolic Ca2+, and ion
channels have been suggested to participate in the transduction from the perception of salinity to the alteration of gene expression [1]. Since alterations in turgor occur in response to both salinity and osmotic stress, and osmotic stress brings about no increased accumulation of G6PDH transcript, it is unlikely that turgor (or any other component common to osmotic and salt stress) is the primary signal linking NaCl to increased G6PDH gene expression. So far, only a few genes that specifi-cally respond to NaCl have been reported, and no
cis-element that confers NaCl-specific activation of gene expression has yet been identified. Charac-terization of the promoter regions of the wheat G6PDH genes will help us to identify the cis-ele-ment conferring the NaCl-specific response.
It is possible that the rapid induction of G6PDH plays a role in mediating plant responses to NaCl stress. In alfalfa cells in suspension, tran-scription rates for G6PDH were increased by elici-tor treatment [12]. In wheat, during the first 10 h of treatment with aluminium solution, G6PDH activity increased in aluminium-resistant cultivar, accompanied by an induction of protein synthesis [13]. G6PDH is a key enzyme of the pentose-phos-phate pathway, which could contribute to NaCl response by providing precursors or cofactors for other biosynthetic routes. For instance, NADPH
Fig. 4. Effect of exposure to 5.0% mannitol (A) or 20 mM ABA (B) on the accumulation of G6PDH transcripts. The intensity of signal, from northern hybridization was measured and standardized by hybridization signals of rRNA gene. The stressed-plant mRNA accumulation was expressed relative to that of the non-stressed control for each stress duration. Relative values were calculated with the value for the 0 h-stressed plant taken as 1.0. The ordinate indicates the rate of increment relative to 0 h, and the abscissa indicates the duration of exposure to NaCl solution. Values represent means9S.E. of two or three replicates. Photographs shows northern blots of total RNAs isolated along the same time course from roots of mannitol- (A), or ABA- (B) treated plants.
Acknowledgements
We thank Dr N. Kawakami for his critical advice during to study and Dr H. Tsujimoto for his critical reading of the manuscript. We also express our appreciation for Professor M. Mii (Chiba University) for the use of his osmometer.
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