JXB Advance Access originally published online on August 8, 2003
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Journal of Experimental Botany, Vol. 54, No. 391, pp. 2265-2274,
October 1, 2003
© 2003 Oxford University Press
Cold-specific and light-stimulated expression of a wheat (Triticum aestivum L.) Cor gene Wcor15 encoding a chloroplast-targeted protein
Received 24 March 2003; Accepted 18 June 2003
Laboratory of Plant Genetics, Department of Biological and Environmental Science, Faculty of Agriculture and Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
* To whom correspondence should be addressed. Fax: +81 78 803 5858. E-mail: nakamura{at}kobe-u.ac.jp
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Abstract |
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Wcor15, a member of the wheat cold-responsive (Cor) gene family, has been isolated and characterized. The deduced polypeptide WCOR15 (MW=14.7 kDa) showed high homology to the previously identified wheat and barley COR proteins. Southern blot analysis using diploid, tetraploid and hexaploid wheat and diploid Aegilops species showed that the wheat and related wild genomes possessed multiple copies of Wcor15 homologues. Five copies were assigned to the homoeologous group 2 chromosomes by nulli-tetrasomic analysis. Northern blot analysis showed that expression of Wcor15 was specifically induced by low-temperature. Homologous transcripts accumulated in leaves, and light markedly increased their steady-state levels. Bombardment-mediated transient expression analysis of a chimeric CaMV 35S::Wcor15-GFP construct showed protein-targeting to epidermal guard cell chloroplasts in excised spiderwort leaves. A promoter of Wcor15 contained at least three CRT/DRE-like sequence motifs found in Arabidopsis Cor genes and induced the reporter GUS gene expression in leaves of transgenic tobacco plants under low-temperature and light conditions. These results suggest that the functional Cor gene system involving the CRT/DRE cis-element is conserved in both monocotyledonous and dicotyledonous plants.
Key words: Cold-responsive (Cor) gene family, chloroplast-targeting, CRT/DRE element, heterologous gene expression, Triticum aestivum L.
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Introduction |
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Cold/freezing temperature is one of the most significant abiotic stresses restricting habitats of sessile plants and reducing crop productivity. Plants in temperate regions have evolved different degrees of ability to survive cold/freezing temperature stress. One prominent adaptive mechanism to this temperature stress is known as cold hardening or acclimation (Levitt, 1980). Cold acclimation is triggered by the exposure of plants to low but non-freezing temperatures for certain periods of time. During this process, plants exhibit dramatic alterations in their gene expression profiles, which are characterized by the induction of a battery of cold-responsive (Cor) genes (Guy et al., 1985; Guy, 1990). Importantly, this adaptive process is believed to be tightly associated with the development of cold/freezing tolerance (Thomashow, 1998, 1999).
Wheat and its relatives, which grow under widely different climatic conditions, exhibit a large genetic variability in cold/freezing tolerance (Fowler and Gusta, 1979; Veisz and Sutka, 1990). A number of genes classified in the families of Lea (late embryogenesis abundant)/Dhn (dehydrin)/Rab (responsive to abscisic acid) have been isolated and their mode of expression has been characterized in wheat, barley and rye. However, the molecular structure and the function of their promoter sequences are largely unknown and, in fact, such important information is only available for the two genes, Wcs120 of wheat (Houde et al., 1992; Vazquez-Tello et al., 1998; Quellet et al., 1998) and Blt4.9 of barley (Dunn et al., 1998). By contrast, significant research results are available for the dicotyledonous model plant Arabidopsis and it has now become a general view that the Cor genes are regulated through a specific signal transduction pathway. A functional cis-acting element of the Arabidopsis Cor genes, i.e. the CCGAC core motif known as a CRT(C repeat)/DRE (dehydration responsive element) sequence, has been proved to play a pivotal role in the promoter function of COR15A/RD29A genes (Yamaguchi-Shinozaki and Shinozaki, 1994; Baker et al., 1994). It has also been shown that the constitutive expression of the CRT/DRE-binding protein genes (CBF/DREB1) in transgenic plants results in the expression of the CRT/DRE-controlled Cor genes without a low temperature treatment and increases their freezing tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998). The wheat Dhn gene Wcs120, which is induced primarily by low temperature (Houde et al., 1992), was also shown to possess two putative CRT/DRE-like motifs in its promoter region (Vazquez-Tello et al., 1998). Furthermore, the promoter of Wcs120 was shown to be cold-inducible in both monocotyledonous and dicotyledonous transgenic plants (Quellet et al., 1998).
Wheat and barley possess a small family of Cor genes including Wcs19 (Chauvin et al., 1993), Wcor14 (Tsvetanov et al., 2000) and Bcor14b (Cattivelli and Bartels, 1990; Crosatti et al., 1999), all of which encode chloroplast-targeted COR proteins analogous to the Arabidopsis protein COR15a (Lin and Thomashow, 1992; Thomashow, 1994). A wheat Cor gene Wcor15, which encodes a chloroplast-targeted protein and shares low-temperature specificity with the barley and wheat Cor genes, has now been isolated. Wcor15 possesses the conserved CRT/DRE-like sequence motifs similar to Wcs120, although their promoter sequences are highly non-homologous. It is further shown that the promoter sequence of Wcor15 renders cold and light responsiveness to transgenic tobacco plants. Taken together, these results suggest that the functional Cor gene system involving CRT/DRE is conserved in both dicotyledonous and monocotyledonous plants.
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Materials and methods |
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Plant materials
Two cultivars of hexaploid (common) wheat (Triticum aestivum L., genome constitution of AABBDD), a winter-type ‘Mironovskaya 808' (abbreviated as M808) and a spring-type ‘Chinese Spring’ (CS), were grown in a controlled-climate cabinet at 25 °C with a 16 h photoperiod at a light intensity of 110–120 µm photons m–2 s–1 provided by cool white fluorescent lamps (the standard temperature and light conditions). M808 was bred in Mironovskaya Institute, Ukraine, and reported to be the hardiest winter cultivar among tetraploid and hexaploid wheat accessions tested for freezing tolerance (Veisz and Sutka, 1990). A single accession each of tetraploid (T. durum cv. ‘Langdon’, AABB) and diploid (T. monococcum, AA) wheat and 9 wild diploid Aegilops species (see Results) were also used in Southern blot analysis for studying the copy number and genome distribution of Wcor15 and Wcor14 genes. A nulli-tetrasomic series of CS (Sears, 1966) was used for chromosome assignment. Each line of the nulli-tetrasomic series lacks a given pair of homoeologous A, B or D genome chromosomes (nullisomic condition) that are replaced by the corresponding homoeologous chromosome pair (tetrasomic condition).
Cloning and sequencing
Leaves of 2-week-old M808 seedlings were harvested and used for the preparation of a nuclear fraction, from which DNA was extracted for the construction of a genomic library. The purified nuclear DNA was partially digested with EcoRI and inserted into the EcoRI site of the phage vector 
gt11 (Stratagene). The initial aim was to obtain genomic clones of the cold-responsive gene Wcor14, and so the genomic library was screened using the Wcor14 cDNA clone as a probe. A genomic clone that was selected through plaque hybridization according to the method previously described (Tsvetanov et al., 2000) was subcloned into pBluescriptII SK–. The sequence was determined using the automated fluorescent dye deoxy terminator cycle sequencing system using ABI PRISMTM 310 Genetic Analyser (PE Applied Biosystems), and the gene was designated as Wcor15 (DDBJ accession number AB095006
[GenBank]
).
Based on the sequence of the isolated Wcor15 genomic clone, the following primer set was designed to isolate cDNA clones containing a complete open-reading-frame (ORF); 5'-ACAACCTA CCCTACCCTACC-3' and 5'-TGTCAGAAAATAAATGCAGC-3'. cDNA clones corresponding to the isolated genomic clone were amplified using total RNA extracted from the cold-acclimated M808 seedlings as templates in the reverse transcriptase PCR (RT-PCR). The amplified fragments were subcloned into pGEM-T vector (Promega, USA) and their sequences were determined. The genomic and cDNA sequences and the deduced amino acid sequences were analysed by DNASIS (Hitachi, Japan). The sequence homology was searched with the BLAST algorithm (Karlin and Altschul, 1993) and a multiple alignment was calculated by Waterman’s algorithm (Waterman, 1986).
Southern and northern blot analyses
Samples of total DNA extracted from diploid, tetraploid and hexaploid wheat, wild diploid Aegilops species and the nulli-tetrasomic series of CS were single-digested with BamHI, XbaI, HindIII and DraI. The digested DNA was fractionated by electrophoresis through 0.8% agarose gel and transferred to Hybond N+ nylon membranes (Amersham). The Southern blots were hybridized with the 32P-labelled genomic clone of Wcor15 and the cDNA clone of Wcor14 as probes. Washing conditions were 2x SSC and 0.1% SDS followed by 0.1x SSC and 0.1% SDS. Probe labelling, hybridization and autoradiography were performed according to Liu et al. (1990).
For RNA extraction, seedlings of M808 and CS were grown for 7 d under the standard temperature conditions in pots with soil. Conditions for cold acclimation were according to Ohno et al. (2001). Briefly, 7-d-old seedlings were placed at 4±0.5 °C under the standard and different light/dark regimes or under continuous dark for different periods. Seven-day-old seedlings were also treated by spraying with a solution containing 20 µM abscisic acid (ABA), 20 µM gibberellic acid (GA3) or 0.4 M NaCl for 1 h, or desiccated on dry filter papers in Petri dishes for 4 h. Total RNA was extracted by guanidine thiocyanate from the above-ground tissues and also from the seedling leaves and roots separately. RNA (20 µg) was fractionated by electrophoresis through 1.2% formaldehyde/agarose gel and transferred to Hybond N+ nylon membranes (Amersham). RNA blots were hybridized with the 32P-labelled whole sequences of the Wcor15 and Wcor14 cDNA clones as probes. Probe labelling, hybridization, washing, and autoradiography were performed in the same way as for Southern blot analysis.
Analysis of chloroplast targeting
The cauliflower mosaic virus (CaMV) 35S promoter of pBI101 (Clontech) was ligated to produce 35S::Wcor15-GFP. A modified GFP, sGFP(S65T) (Chiu et al., 1996), was used and the chimeric construct was cloned into competent JM109 cells (TOYOBO). After structural confirmation of the construct, they were introduced by particle bombardment according to Takumi et al. (1994) into the dorsal side of epidermal cell layers peeled off from excised spiderwort (Tradescantia reflexa) leaves. The autofluorescence (red) and the GFP images of chloroplasts were observed under fluorescence light microscopy (Olympus).
Transgene construction, tobacco transformation and GUS assay
The 5' upstream sequence of Wcor15 amplified from the selected genomic clone was replaced by the CaMV 35S promoter of pBI101 (Clontech) to produce the Wcor15::GUS construct. Transgenic tobacco plants were produced by the Agrobacterium-infection method. The GUS construct under control of the Wcor15 promoter was introduced into leaf discs of Nicotiana tabacum cv. ‘Petit Havana’ using Agrobacterium tumefaciens LBA4404. Transformants were selected in MS medium (Murashige and Skoog, 1962) containing 0.1 mg l–1 NAA, 1.0 mg l–1 BA and 250 mg l–1 kanamycin. The transformants (T0 generation) were regenerated on hormone-free MS medium containing 50 mg l–1 kanamycin.
GUS activity was assessed histochemically using the kanamycin-resistant homozygous T2 progeny. The T2 progeny plants were grown for 7 d in pots with soil at 27 °C under a 16 h photoperiod at a light intensity of 110–120 µmol photons m–2 s–1 provided by cool white fluorescent lamps. The plants were then cold acclimated for 7 d at 4±0.5 °C under the same photo-intensity and photoperiod conditions. A staining solution for the GUS assay contained the following components: 1.9 mM 5-bromo-4 chloro-3-indoyl-ß-D-glucuronic acid (X-gluc), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.3% Triton X-100. The transgenic tobacco plants were incubated with the staining solution at 37 °C for 24 h.
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Results |
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Cloning of genomic and cDNA sequences of Wcor15
Using the M808 genomic library and the Wcor14 cDNA probe, one lambda phage clone was selected from c. 150 000 pfu and subcloned into the HindIII site of the vector pBluescriptII SK–. BLAST analysis showed that this 4.2 kb subclone contained an ORF with varying degrees of homology to the previously reported wheat and barely Cor genes, i.e. wheat Wcs19 (Chauvin et al., 1993) and Wcor14 (Tsvetanov et al., 2000) and barley Bcor14b (originally designated as pt59) (Cattivelli and Bartels, 1990). After RT-PCR using RNA templates extracted from the cold-acclimated M808 seedlings, one cDNA sequence showing a high homology to Wcs19 was identified. This cDNA clone had an ORF of 441 nucleotides and putatively encoded a polypeptide with 147 amino acid residues (Fig. 1). Comparing the cDNA sequence with the genomic sequence, the gene was found to contain two exons with an intron of 108 bp in length. The deduced polypeptide was acidic (pI=5.0) and hydrophilic with a molecular weight of c. 14.7 kDa. Since the basic exon/intron structure in Wcor14 was conserved in this sequence, the gene was designated Wcor15.
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Amino acid sequence of the deduced polypeptide WCOR15 showed 97.2% identity with that of the previously identified and recently revised chloroplast-targeted wheat WCS19 protein (Chauvin et al., 1993; NDong et al., 2002) (Fig. 1). This suggested that these represented either homoeologous or paralogous wheat COR proteins. On the other hand, WCOR15 showed a limited amino acid identity (less than 43.2%) with another acidic and hydrophillic wheat WCOR14 (Tsvetanov et al., 2000) and barley BCOR14b (Cattivelli and Bartels, 1990). All of these COR proteins shared the almost identical N-terminal 50 or 51 amino acid residues, which were predicted to be a chloroplast-targeting signal peptide (Chauvin et al., 1993; Gray et al., 1997; Crosatti et al., 1995, 1999). The intron insertion site in Wcor15 was just after the putative chloroplast transit peptide (Fig. 1). The intron insertion site of Wcor15 was conserved in Wcor14 (Tsvetanov et al., 2000), while the nucleotide sequence of the Wcor15 intron showed no homology with that of Wcor14.
Copy number estimation and chromosome assignment
To study copy number and genome distribution of the Wcor15 gene in the wheat genome, in comparison with that of Wcor14, Southern blot analysis was conducted using DNA extracted from a single accession each of hexaploid, tetraploid and diploid wheat. The Southern blot patterns probed with the Wcor15 genomic clone were different from those probed with the Wcor14 cDNA clone (only the patterns of Wcor15 are shown in Fig. 2A), suggesting that they did not cross-hybridize and were located at different positions on the wheat genomes. The Southern blots showed that Wcor15 and its homologous sequences comprised a small multigene family in the diploid (AA genome), tetraploid (AABB genome) and hexaploid (AABBDD) wheat. A chromosome assignment was then made using DNA extracted from the nulli-tetrasomic series of CS. This deletion/duplication analysis showed that at least five major homologues of Wcor15 were located on the homoeologous group 2 chromosomes in the hexaploid wheat genome (Fig. 2B). A and B genomes contained at least two copies. Wcor14 was also assigned to the homoeologous group 2 chromosomes (data not shown).
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Because of the small multigene nature of Wcor15 in the wheat genomes, their copy number and genome distribution were studied further in nine diploid species of Triticum and Aegilops (Fig. 2C). Southern blot analysis clearly showed that all of these species possessed more than two homologous copies in their diploid genomes. It was further noted that different accessions within a given species showed restriction fragment length polymorphisms, indicating that the copy number and genomic distribution of Wcor15 homologues varied in the wheat and related wild genomes. The variation was largest in Ae. speltoides, a putative B genome donor to tetraploid and hexaploid wheat.
Effect of cold treatment, dehydration stresses and plant hormones on the steady-state transcript levels
The gene expression of Wcor15 was monitored and compared with that of Wcor14 using M808 and CS seedlings. Since these two wheat Cor genes showed different patterns in the Southern blots, it was considered that they did not cross-hybridize to the northern blots under the high stringency washing condition employed. However, it was assumed that the northern blot data probably represented mixtures of these and possibly other homologous transcripts, including that of Wcs19. No homologous transcripts were detected in the above-ground tissues of the non-acclimated control seedlings (Fig. 3A). Under the low temperature condition with the standard 16/8 h light/dark photoperiod, both of the Wcor14 and Wcor15 homologous transcripts showed high levels of accumulation in the seedling leaves after 3–5 d. The cold-inducible gene expression was detected within 4 h in M808, while it was delayed until after 6 h in CS (Fig. 3B). A clear cultivar difference was observed at 12 h: M808 accumulated more transcripts than CS. The effect of light on gene expression was studied next. The longer period of continuous dark treatment markedly reduced the amount of both Wcor14 and Wcor15 homologous transcripts (Fig. 3A). The reduction was more prominent in Wcor15 than in Wcor14 in both cultivars. An additional 3 d acclimation in the dark after 5 d acclimation in the standard light condition (5L3D in Fig. 3A) did not affect the transcript levels, suggesting that the transcripts were stable for at least 3 d under this condition. An additional 3 d acclimation in the standard light condition after 5 d acclimation under the dark (5D3L), however, markedly increased the transcript levels of both genes. The result suggested that transcription of these Cor genes were enhanced by light and suppressed by darkness. M808 showed a higher stimulation by light under the low-temperature condition than CS. Because of the light stimulation, the level of transcript accumulation in the seedling leaves and roots were compared separately. It was shown that the accumulation of both transcripts was restricted to the seedling leaves (Fig. 3C). No homologous transcripts were detected in the seedlings treated with 400 mM NaCl, 20 µM ABA and GA3 and dehydration under the standard temperature condition (data not shown). It was thus clearly shown that the expression of Wcor15 homologues and Wcor14 was low-temperature-specific and ABA-independent in the both cultivars.
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GFP assay for chloroplast targeting
The leaf-specific expression and the highly conserved structure of the putative chloroplast transit peptides at the N-terminal strongly suggested that WCOR15 homologous proteins as well as WCOR14 are targeted and function in chloroplasts, similar to the barley BCOR14b and wheat WCS19. Chloroplast-targeting of BCOR14b and WCS19 was previously shown either by Western blot analysis of the chloroplast protein fraction (Crosatti et al., 1999) or immuno-localization by electron microscopy of the purified chloroplasts (NDong et al., 2002). To prove the chloroplast-targeting of WCOR15, a transient expression system using a chimeric CaMV 35S::Wcor15-GFP construct introduced by particle bombardment into the lower (dorsal side of) epidermal cell layers peeled off from excised spiderwort leaves was employed. The transient expression of the GFP fusion protein clearly demonstrated targeting of WCOR15 to the epidermal guard cell chloroplasts (Fig. 4). It was also noted that GFP signals were observed in the epidermal cells other than the guard cells. Since no clear red autofluorescence image was observed in epidermal cells, the observation suggested that WCOR15 could also be targeted into the epidermal proplastids under the transient and heterologous expression system.
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Cold-responsiveness of the promoter in transgenic tobacco leaves
In the Wcor15 gene, a TATA-box was located at the 102 bp upstream position of the translation initiation site. Four CRT/DRE-like sequences containing the CCGAC core motif were identified within the 2 kb upstream region of the Wcor15 ORF. To examine if the cold-responsiveness of the Wcor15 gene is transcriptionally controlled by the 5' upstream regulatory sequence, a transgenic experiment using tobacco plants was conducted. A chimeric gene was constructed in that a GUS reporter gene was placed under the control of the 1.7 kb upstream sequence of Wcor15 containing the three downstream CRT/DRE motifs (Fig. 5A). The Wcor15::GUS construct was introduced into the tobacco genome through the Agrobacterium-infection method. Kanamycin-resistant progeny plants (T1 generation) were selected from self-pollinated seeds of the regenerated transgenic tobacco plants (T0 generation), in which the Wcor15::GUS integration was confirmed by Southern blot analysis (data not shown). A T2 homozygous population for the introduced GUS gene was selected from the kanamycin-resistant T1 plants. The T2 seedlings were grown under the condition of 16 h day-length and 27 °C for 7 d, and then treated with low temperature (4±0.5 °C) under 24 h light or dark for 7 d. Remarkable GUS expression in the leaves was observed only in the low-temperature-treated seedlings under the light condition (Fig. 5B). Under the dark condition, the low-temperature treatment resulted in slightly detectable levels of GUS accumulation, while the non-treated transgenic plants showed no detectable signals in the leaves and a very low level in the stems. No GUS signals were detected in the non-photosynthetic root tissues of the transgenic plants, irrespective of the temperature and light conditions. The cold-responsiveness of the Wcor15 gene expression, therefore, was successfully demonstrated in the heterologous tobacco system.
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Discussion |
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Wcor15, a member of the Cor gene family, has been isolated from the winter-hardy common wheat cultivar M808. The deduced protein WCOR15 showed a highly identical amino acid sequence with that of the wheat WCS19 (Chauvin et al., 1993; NDong et al., 2002) (Fig. 1). This high structural similarity indicates that they are either homoeologues or paralogues in the allopolyploid wheat genome. Genomic Southern blot analysis using diploid, tetraploid and hexaploid wheat, in fact, showed that Wcor15 comprised a small multigene family (Fig. 2). At least five copies were located on the homoeologous group 2 chromosomes in hexaploid wheat by the nulli-tetrasomic analysis. Although chromosomal assignment of Wcs19 has yet to be made (NDong et al., 2002), it also probably locates on the group 2 chromosomes. The deduced WCOR15 protein, on the other hand, showed much lower levels of homology with two other COR proteins, i.e. the barley BCOR14b (Cattivelli and Bartels, 1990) and its wheat orthologue WCOR14 (Tsvetanov et al., 2000). The Wcor14 gene was located on the group 2 chromosomes in this study (data not shown). Similarly, Bcor14b was located on the barley chromosome 2H (Cattivelli et al., 2002), which is homologous to the group 2 chromosomes of wheat (Linde-Laursen et al., 1997). Southern blot profiles of Wcor15 and Wcor14, however, were different, suggesting that the chromosomal location of these two wheat Cor genes are different and thus they are paralogues possibly generated by duplication in the ancestral diploid genomes. The conserved intron insertion site (Fig. 1), but different intron and second exon sequences, further suggest that Wcor15 and Wcor14 represent products of exon shuffling. Significant variations were further revealed among the wild and cultivated diploid Triticum and Aegilops genomes. The result clearly suggests that the gene duplication and diversification occurred at the diploid level. The largest variation was detected among accessions of Ae. speltoides, a putative B genome donor to tetraploid and hexaploid wheat. A further comparative analysis aiming at evaluating the diversity in the Wcor15 gene family has to be conducted.
Expression of the Wcor15 gene family and its paralogous Wcor14 was specifically induced by low temperature and apparently stimulated by light (Fig. 3). In the previous study, the developmental time-course of the freezing tolerance and that of the transcript accumulation of two cold-inducible genes Wlt10 and Wdhn13 were compared in two wheat cultivars, a winter-type M808 and a spring-type CS (Ohno et al., 2001, 2003). Under the bioassay conditions, the level of freezing tolerance increased dramatically and reached high plateau levels at 3–5 d after cold acclimation at 4 °C in both cultivars. A good correlation was observed between the levels of transcript accumulation and the levels of freezing tolerance in the two cultivars during 10 d of cold acclimation. M808 developed a much higher level of freezing tolerance and accumulated more transcripts than CS. The time-course of accumulation of the Wcor14 and Wcor15 transcripts in the present study (Fig. 3) was similar to that of Wlt10 and Wdhn13. M808 accumulated transcripts more rapidly than CS. It has already been shown that the level of expression of Bcor14b, a barley orthologue of Wcor14, was tightly linked to freezing tolerance and so cultivar differences were explained at the transcriptional level (Vagujfalvi et al., 2000). The constitutive expression of the Wcs19 gene in transgenic Arabidopsis leaves using the CaMV 35S promoter was also shown to increase the level of freezing tolerance, although only when a low temperature treatment was given (NDong et al., 2002). Taken together, these results strongly suggest that the Cor and related genes play an important role in cold/freezing tolerance in wheat and barley.
WCOR15 was shown to be targeted into the epidermal guard cell chloroplasts in the transient assay system (Fig. 4). Recently, NDong et al. (2002) identified by various ways, including cDNA library selection, PCR amplification and data mining, five additional Cor-related sequences that putatively encode chloroplast-targeted proteins from wheat, barley and rye. Although expression patterns of these Cor genes were not studied, they were classified into three groups of an Lea3-like family based on their common protein structures. According to this classification, Wcor14 and Bcor14b belong to the same group L1 and Wcor15 and Wcs19 belong to another group L2. Apparently these Cor genes comprise a gene family with similar function. It is thus suggested that the expression of the wheat, barley and rye Cor genes, whose protein products are transported into chloroplast, are regulated through the same or at least partly overlapped signal transduction pathways. Importantly, all of these wheat and barley Cor genes are regulated by a seemingly complex interaction of low-temperature and light (Gray et al., 1997; Crosatti et al., 1999). It is generally known that low temperatures typically reduce membrane integrity (Nishida and Murata, 1996; Thomashow, 1999) and adversely affect photosynthetic electron transport and carbon fixation in chloroplasts (Guy, 1990; Huner et al., 1998). Under subzero temperature conditions, the electron transport chain tends to be over-reduced (Huner et al., 1998), resulting in photoinhibition of the photosystem II (PSII) and increased production of reactive oxygen species (Asada, 1994). It was shown that the level of Wcs19 mRNA accumulation was correlated with that of the PSII excitation pressure (Gray et al., 1997). These results suggest that expression of the wheat and barley Cor genes and accumulation of the COR proteins help maintain the activity of the photosynthetic apparatus through regulation of the redox state in chloroplasts under subzero temperatures.
The Arabidopsis genome contains analogous gene Cor15a that encodes a chloroplast-targeted COR protein and is regulated through the interaction between the CRT/DRE cis elements and the CBF/DREB1 transcription factors (Yamaguchi-Shinozaki and Shinozaki, 1994; Baker et al., 1994). It was found that the Wcor15 gene contains four CRT/DRE-like motifs in its promoter region. Furthermore, it was shown that the native Wcor15 promoter containing the three downstream CRT/DRE motifs was cold- and light-responsive in leaves of the transgenic tobacco plants (Fig. 5). It is noted that the promoter of Wcs120, a cold-specific member of the wheat Dhn genes locating on the group 6 chromosomes (Limin et al. 1997), contains two CRT/DRE-like sequences in the regions showing cold responsiveness and it is functional in both monocotyledonous and dicotyledonous transgenic plants (Quellet et al. 1998). Since no clear homology was found between the overall promoter sequences of Wcor15 and Wcs120, the shared short cis-elements of CRT/DRE-like sequences might be the primary determinant of the cold-responsive expression at least in the Wcor15 and Wcs120 genes. The result that the CRT/DRE motifs regulated the Wcor15 gene expression at the transcriptional level and precisely functioned in the heterologous tobacco system strongly supports this contention. Although a final conclusion has to wait until a more direct result of the promoter function is obtained, this present result that the Wcor15 promoter correctly responded to the combination of low-temperature and light in cold-sensitive tobacco plants suggest the functional role of the CRT/DRE-like motifs both in the monocotyledonous wheat and dicotyledonous tobacco. Since the barley Cor gene Blt4.9 is known to possess a non-CRT/DRE-dependent promoter (Dunn et al., 1998), there is an urgent need to determine if the wheat Wcor14 and Wcs19 and barley Bcor14b genes share the CRT/DRE-like motifs in their promoters. Further studies are also required to examine the functional interaction of these cis elements with CBF/DREB1-orthologous transcription factors in wheat, barley and other cereal species.
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Acknowledgements |
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We thank Dr Y Niwa, Shizuoka University, Japan, for his gift of the sGFP(S65T). The work was supported by a ‘grant-in-aid’ from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to CN, no. 13306002). The sequence of Wcor15 has been deposited in the DDBJ database (accession number AB095006 [GenBank] ). This paper is contribution no. 153 from the Laboratory of Plant Genetics, Faculty of Agriculture, Kobe University.
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