A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar 'Mironovska 808

Genes Genet. Syst. (2000) 75, p. 49–57 A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar ‘Mironovska 808’ Sergei Tsvetanov1, Ryoko Ohno1, Kanako Tsuda1, Shigeo Takumi1, Naoki Mori1 Atanas Atanassov2 and Chiharu Nakamura1,* 1 Department of Life Science, Graduate School of Science & Technology, and Laboratory of Plant Genetics, Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan 2 Institute of Genetic Engineering, 2232 Kostinbrod, Bulgaria (Received 20 December 1999, accepted 21 February 2000) A cDNA library was constructed from a cold-acclimated winter-hardy common wheat (Triticum aestivum L.) cultivar ‘Mironovska 808’. Using this library and a cold- and light-responsive barley cDNA clone cor14b as a probe, cDNAs of a homologous wheat gene wcor14 were isolated. Two identical cDNAs designated as wcor14a had an open reading frame encoding an acidic (pI = 4.71) and hydrophobic polypeptide with 140 amino acids (MW=13.5 kDa). The deduced WCOR14a polypeptide showed 70% identity with the barley chloroplast-imported COR14b and had a nearly identical N-terminal, putative chloroplast transit peptide of 51 amino acid residues. Another cDNA clone wcor14b was assumed to encode a polypeptide WCORb which had 5 substitutions and a frame shift in the C-terminal region as compared with WCOR14a. RACE PCR, genomic PCR and Southern blot analyses suggested that wcor14 and its related sequences constitute a small multigene family with and without an intron in the hexaploid wheat genome. Northern blot analysis showed that transcripts of wcor14 accumulated within 3–6 hours of cold acclimation at 4°C and the level reached a maximum at day 3. The transcripts became non-detectable within 3 hours after de-acclimation at room temperature. Contrary to the barley cor14b, a similar level of wcor14 transcripts was detected under the continuous darkness. Neither treatment with NaCl, ABA nor dehydration induced its expression. Based on these results we conclude that wcor14 is a wheat orthologue of the barley cor14b and specifically induced by low temperature. INTRODUCTION Herbaceous plants growing in the temperate regions have ability to develop tolerance to freezing temperature after some periods of exposure to low but nonfreezing temperature. During this adaptive process of cold acclimation or cold hardening, a range of physiological and biochemical changes take place in vegetative tissues in plants (Graham and Patterson, 1982; Guy, 1990; Li and Christersson, 1993). Since the first discovery of significant changes in gene expression during the cold acclimation process in plants (Guy et al., 1985), much efforts have been devoted to understand the molecular basis of this process (Guy, 1990; Thomashow, 1990, 1994). According Edited by Kiyotaka Okada * Corresponding author. E-mail: nakamura@kobe-u.ac.jp to Thomashow (1994, 1998), cor (designated after cold-responsive or cold-regulated) genes are groups of genes which encode extremely hydrophillic and boiling-stable COR proteins. Dehydrins (DHN)/late embryogenesis abundant proteins (LEA)/proteins responsive to abscisic acid (RAB) are the well known classes of COR proteins. Some COR proteins, like Arabidopsis KIN1, show structural similarities to antifreeze proteins (Kurkela and Franck, 1990) and others to a HSP70 chaperon family which is known to have roles in protein transport, folding and assembly/disassembly processes (Neven et al., 1992). Although physiological function of these different classes of COR proteins remains largely unknown, some showed a positive correlation with the degree of freezing tolerance which is enhanced by cold acclimation (Monroy et al., 1993; Houde et al., 1995; Jaglo-Ottosen et al., 1998). In wheat two COR-DHN subfamilies have been 50 Sergei Tsvetanov et al. identified. A gene wcs120 and its related, perhaps paralogous genes including Wcor39, Wcs66, Wcor80, Wcor200 and Wcor726 are expressed in response to low temperature (Houde et al., 1992; Guo et al., 1992; Quellet et al., 1993; Chauvin et al., 1994; Danyluk et al., 1996; Hughes and Dunn, 1996). Among them, wcs120 and wcor200 are the most well characterized members respectively encoding glycine-rich neutral proteins of 50 kDa (pI = 7.3) and 200 kDa (pI = 6.5) with characteristics of the group 2 LEA (or D-11) family, although they do not have a conserved polyserine sequence (Houde et al., 1992; Quellet et al., 1993). wcor410 encodes an acidic (pI = 5.1) DHNlike group 2 LEA protein of 28 kDa with 15-mer lysinerich motifs and a polyserine sequence (Danyluk et al., 1994). Tarc7 is another low-temperature specific wheat gene not belonging to the dhn family (Gana et al., 1997). Barley cor14b is a unique member of the cereal cor gene families encoding an acidic protein designated as COR14b (Cattivelli and Bartel, 1990). COR14b is, like Arabidopsis thaliana COR15a (Lin and Thomashow, 1992; Thomashow, 1994), a leaf-specific protein and transported into the stromal compartment of the chloroplasts during cold acclimation (Crosatti et al., 1995, 1999). In contrast to Arabidopsis COR15a, however, barley COR14b is considerably hydrophobic and its expression is light-stimulated (Crosatti et al., 1995, 1999). In wheat, one unique member of cor genes designated as wcs19 encodes a leafspecific and basic (pI = 8.8) protein WCS19 which is also transported into the stromal compartment of the chloroplasts during cold acclimation (Chauvin et al., 1993; Gray et al., 1997). Since WCS19 shows a low level of homology with the barley COR14b, we attempted to isolate the wheat orthologue of cor14b. We expected that the hexaploid wheat genome has at least triplicate homoeologous loci of this gene. We herein report the structural heterogeneity of cDNAs, genomic sequences and distribution, and low temperature-specificity of the identified wheat gene wcor14. MATERIALS AND METHODS Plant materials. A winter-hardy common wheat (Triticum aestivum L.) cv. ‘Mironovska 808’ (abbreviated as M808), a spring-type common wheat cv. ‘Chinese Spring (CS), two durum wheat cultivars (T. durum Desf. cv. ‘ Langdon’ and T. turgidum L. var. reichenbachii), a cultivated timopheevi wheat (T. timopheevi var. typicum), and a diploid wheat (T. monococcum L. var. vulgare) were used. Growing conditions of these plants are described in the following sections. Construction of a cold-acclimated cDNA library and cloning and sequencing of wcor14 cDNAs. A winterhardy common wheat cv. M808 was bred in Mironovska Institute in Ukraine. It was reported as the hardiest cul- tivars among tetraploid and hexaploid wheat tested for freezing tolerance (Veisz and Sutka, 1990). M808 therefore was selected for the construction of a cold-acclimated cDNA library. Seedlings (23-day-old) of M808 were coldacclimated for 9 days at 4 ± 0.5°C under the standard light condition at an intensity of 55 to 65 µmol・m–2・s–1 provided by cool white fluorescent lumps with a 16 hour photoperiod. The leaves were harvested at the end of this period and used for the cDNA library construction. Poly(A)+-RNA was purified by two passages of total RNA through an oligo-dT cellulose column (Pharmacia Biotech). The first and second strands were synthesized using Pharmacia Biotech cDNA Synthesis Kit. The double-stranded cDNAs were cloned into the EcoRI site of the phage vector λgt10 (Stratagene) and transformed into an Escherichia coli strain NM514. The cDNA library was screened using a cold-responsive barley cDNA clone, cor14b (originally designated as pt59) (Cattivelli and Bartel, 1990), as a probe. cor14b was provided by the authors. Plaque screening was made in 50 % formamide, 6 × SSC, 5 × Denhardt’s solution, 0.5 % SDS, and 20 mg・ ml-1 salmon sperm DNA at 42°C for 14 h. Nylon membranes (HybondTM–N+, Amersham) were washed in 2 × SSC containing 0.1 % SDS at room temperature and further washed in 0.2 × SSC containing 0.1% SDS at 65°C. Probe labeling was carried out using ECLTM Direct Nucleic Acid Labeling and Detection System (Amersham). Sequencing was carried out according to Sanger’s dideoxynucleotide chain-termination method using SequiTherm EXCELTM II Long-ReadTM DNA sequencing Kit-LC (Epicentre Technologies). RACE PCR analysis. Total RNA was isolated from 3day cold-acclimated M808 plants (17-day-old) and purified using a mRNA Purification Kit (Pharmacia). To obtain full-length cDNAs, 5’- and 3’-RACE PCRs were performed using Advantage cDNA PCR Kit (CLONETECH). For 5’RACE PCR the first-strand cDNA was synthesized from 1 µg of poly(A)+-RNA and a wcor14-specific primer RA (5’AAGCACGGCCTGGGAAGAGC-3’) (for the primer location, see Fig. 1). After the second stand synthesis, PCR was conducted using this primer and an adapter primer. In a 3’-RACE PCR system, the first-strand cDNA was synthesized using a polyT primer and the 3’ part was amplified using a wcor14-specific primer RB (5’CTGCCTGCAAACCCCTCCTA-3’) and the same adapter primer. Amplified fragments were cloned into the pGEM-T vector (Promega) according to the manufacturer’s instruction. After introduction of these recombinant plasmids into E. coli JM109, clones were randomly selected and sequenced by Sanger’s dideoxynucleotide chaintermination method as described. Genomic PCR analysis. Total DNA was extracted from M808 and used for PCR amplification of the genomic A cold-responsive wheat gene wcor14 51 Fig. 1. Nucleotide and deduced amino acid sequences of the cold-responsive wheat cDNA, wcor14a. The start and stop codons were indicated by underlined bold-faced letters. An intron position was boxed with an asterisk. The positions of primers used for RACE PCR (RA and RB) and genomic PCR analyses (U1-L1 and U2-L2) were indicated by underlines. wcor14 sequence. A nested-PCR was conducted in the following way: the first amplification was performed using an upper primer U1 (5’-CTCGTCCCCACACCGTCAGC) and a lower primer L1 (5’-TTGCTCACATCCTCGACCGC) and the second amplification using a primer U2 (5’-CTGCCTGCAAACCCCTCCTA) and a primer L2 (5’-CCTCCTCCGTCGCCTGCTTCGCCT) (for the primer locations, see Fig. 1). A reaction mixture contained 1.5 mM MgCl2, 0.1 mM dNTPs and 2.5 U Taq polymerase (Takara) and the amplification reaction was carried out in 25 cycles of 94°C for 30 s, 68°C for 20 s, and 72°C for 2.3 min. The amplified fragments were sepa- 52 Sergei Tsvetanov et al. rated by 13% polyacrylamide gel electrophoresis. They were cut out, individually cloned into pGEM-T vector (Promega) and sequenced as described. Southern blot analysis. Total DNAs were extracted from leaves of 2 to 3-week-old plants of M808 and CS, T. turgidum var. reichenbachii, T. durum cv. ‘Langdon’, T. timopheevi var. typica, and T. monococcum var. vulgare. DNAs were digested with EcoRI, BamHI, HindIII or DraI. Electrophoresis was conducted through 0.8% agarose gel and the gel was blotted onto nylon membranes (Hybond N+, Amersham) by the alkaline blotting method. Probe DNA (wcor14a) was labeled with [α-32P] dCTP using Random Primed DNA Labeling Kit (Boehringer Manheim). Hybridization and detection of the signals were performed according to Liu et al. (1990). Northern blot analysis. Plants of M808 were grown for 2 weeks at 25/20°C under the standard light condition. The plants were then cold acclimated for different periods at 4 ± 0.5°C. After this cold acclimation treatment, some plants were de-acclimated by transferring them back to the normal temperature condition. The plants were also grown for different periods at 4 ± 0.5°C under the continuous darkness. For the treatment with NaCl or abscisic acid (ABA), two-week-old plants were placed in glass tubes containing a 400 mM NaCl solution or a 20 µM ABA solution, and kept for 3 days at 25 ± 0.5°C under the 16L8D photoperiod. For desiccation, the same-aged plants were kept on dry filter paper for 3 hours at 25°C under the light condition before RNA extraction. Total RNA was extracted from the aerial parts of the plants using ISOGEN (Wako). RNA (20 µg) was denatured at 65°C for 5 min in 50% (v/v) formamide, 1 × MOPS (200 mM MOPS, 10 mM EDTA, 50 mM NaOAc, pH 7.0) and 1.5% formaldehyde. The RNA samples were subjected to electrophoresis through 1.2% denaturing agarose gel in the presence of 0.73 M formaldehyde, and blotted onto nylon membranes (HybondTM-N+, Amersham). [α32 P]dCTP-labeled wcor14a was hybridized to RNA blots in 5 × SSC containing 0.1% sarcosyl, 0.02% SDS, 5% blocking reagent (Boehringer Manheim) and 50% formamide at 42°C overnight. Membranes were washed twice in 0.2 × SSC containing 2% SDS at 65°C for 1 hour, exposed to Imaging Plate (Fuji Film) at room temperature for appropriate periods. The signals were analyzed with a Bio Image Analyzer, BAS2000 (Fuji Film). RESULTS AND DISCUSSION Cloning and structural analysis of wcor14 cDNAs and their deduced WCOR14 polypeptides. A period of cold acclimation is an important factor for the construction of the cDNA library suitable for cloning cor genes. An early study on the protein profile in response to low temperature in wheat demonstrated that two groups of translatable mRNAs were expressed during cold acclimation (Danyluk et al., 1991). The first group was transient and consisted of 18 mRNAs that reached their highest levels of induction after 1 day of low temperature exposure but thereafter decreased to undetectable levels. The second group consisted of 53 mRNAs that were also induced rapidly but maintained their high levels of expression during 4 weeks of the experiment. Among the second group, at least 34 were expressed at higher levels in freezing tolerant winter wheat cultivars than in less tolerant spring wheat cultivars. Because of our interest in the second group, we used 9-day cold-acclimated M808 plants for the construction of the cDNA library; the period of which was considered to be sufficient for the isolation of the group 2 cor members. A plaque screening was conducted using the barley coldresponsive cDNA clone cor14b as a probe. Three cDNA clones were obtained after screening of ca. 60,000 plaque-forming units. Two identical clones (designated as wcor14a) had 638 bp containing an open reading frame which was predicted to encode a 13.5 kDa acidic (pI = 4.71) polypeptide of 140 amino acid residues (Fig. 1). The deduced amino acid sequence of this protein (WCOR14a) showed 70% identity with the barley COR14b (pI = 4.5) (Fig. 2). Notably, a stretch of the N-terminal 51 amino acid residues of WCOR14a was nearly identical to that of the barley COR14b (98% identitity with only one amino acid difference), and highly homologous (78% identity) to that of the wheat WCS19 if its reported further upstream sequence is excluded in the comparison. The remaining down stream part of WCOR14a showed 54% identity with COR14b but only 34 % identity with WCS19. In contract to Arabidopsis COR15a, WCOR14a was considerably hydrophobic (59% hydrophobic residues) similar to COR14b and WCS19 (56% and 55%, respectively). The barley COR14b immunologically cross-reacts with a related, chloroplast-imported protein COR14a (Crosatti et al. 1999). N-terminal microsequencing of COR14b was unsuccessful but that of the N-terminal 11 amino acids of COR14a purified from the chloroplast fraction suggested that it was encoded by a gene cor14a independent from cor14b. Because of the homology of this partial N-terminal sequence with the corresponding part of WCS19, the barley cor14a was suggested to be orthologous to the wheat wcs19. Interestingly, the N-terminal 51 amino acid residues were absent in COR14a purified from the chloroplast fraction. This sequence was thus suggested to serve as a chloroplast transit peptide (Crosatti et al. 1999). Similar to the barley COR14b, however, the N-terminal 51 amino acids of WCOR14a lacked the loosely defined consensus cleavage sequence of (Val/Lle)-X-(Ala/ Cys)-Ala which is characteristic for transit peptides that target nuclear-encoded proteins to the stromal compartment of chloroplasts (Gavel and von Heijne, 1990). A cold-responsive wheat gene wcor14 53 Fig. 2. Amino acid alignment of WCOR14a, b and c with the barley COR14b and wheat WCS19. WCOR14a and b were deduced from the corresponding cDNA sequences and WCOR14c from the 3’-RACE PCR product (3’-RACE PCR-2 in Fig. 3). The boxed N-terminal 51 amino acid residues represent a putative chloroplast transit peptide. The underlined amino acids are identical throughout WCOR14a, b and c and COR14b. Except for this discrepancy, the sequence had several features in common with the reported chloroplast transit peptides. First, it had an arginine residue at the position –9 relative to the putative cleavage site at the position 51-52 (Fig. 2). Second, it had a relatively high serine plus threonine content (12%) but had no acidic residues. Third, it had an uncharged N-terminal domain (residues 1 to 23), a central domain (24 to 41) containing 3 positively charged residues and lacking acidic residues (Garnier et al., 1978). Based on these criteria, it was predicted that this N-terminal sequence represented a novel chloroplast transit peptide. Heterogeneity in wcor14 transcripts. It was reasonable to expect that the hexaploid wheat genome has at least triplicate homoeologous wcor14 loci. We in fact iso- lated one additional cDNA clone wcor14b which had 25 nucleotide substitutions and a one-nucleotide deletion in the coding region as compared with wcor14a. This clone had an open reading frame encoding a protein WCOR14b which had 5 amino acid substitutions and a frame shift in the C-terminal region relative to WCOR14a (Fig. 2). To obtain full sequences of wcor14 cDNAs and to further study the possible presence of homoeologous transcripts, RACE PCR analyses were conducted using the primer RA for 5’-RACE PCR and the primer RB for 3’-RACE PCR (for the primer locations, see Fig. 1). Two types of sequences were obtained by 5’-RACE PCR (data not shown). One (5’-RACE PCR-1) had additional 8 nucleotides in the 5’ non-coding region and the remaining part was identical to wcor14a cDNA. The other one (5’-RACE PCR-2) had additional 21 nucleotides and a 17-nucleotide deletion in 54 Sergei Tsvetanov et al. the 5’ non-coding region as compared with wcor14a. This novel transcript had a T nucleotide instead of C at position 149 like wcor14b. Amino acid replacement did not occur by this nucleotide substitution. Two different types of sequences were obtained by 3’-RACE PCR in addition to that corresponded to wcor14a (data not shown). Three clones (3’-RACE PCR-1) had an identical sequence to that of wcor14a but all had an 18-nucleotide shorter 3’ non-coding region with a poly A tail. The other four clones (3’RACE PCR-2) had three nucleotide substitutions together with a one-nucleotide addition and a deletion in the C-terminal coding region. One of these nucleotide substitutions and the one nucleotide addition and deletion resulted in a putative protein WCOR14c with an amino acid replacement at the position 63 and frame shifts in the C-terminal region as compared with its corresponding region of WCOR14a and WCOR14b (Fig. 2). This novel transcript had a polyA tail and a 65-nucleotide shorter 3’ non-coding region than that of wcor14a. Although the number of clones we analyzed was limited, the results suggested that at least four different wcor14 cDNAs including wcor14b are present in the hexaploid genome of M808. Genomic structure of wcor14. To further analyze genomic structure of the wcor14 loci, nested genomic PCR amplification was carried out using primer sets of U1-L1 and U2-L2 (for the primer locations, see Fig. 1). In this study, total DNA extracted from M808 was used as a template. The result showed that there were two different sized introns (174 bp of intron I and 97 bp of intron II) delimited by GT-AG in the wcor14 coding region (Figs. 1 and 3). A sequence without intron was also detected in the M808 genome. The genome specificity of these sequences has to be further clarified. Genomic copy number of wcor14. The barley cor14b was reported to be a single locus gene, and genomic Southern blot analysis of the hexaploid wheat genome using cor14b as a probe suggested the presence of several homologous wheat genes (Cattivelli and Bartel, 1990). Since we could detect two different cDNA sequences of wcor14, at least three homoeologous transcripts by RACE PCR and three genomic sequences by genomic PCR, we next studied the copy number of wcor14 and its related sequences in the wheat genome by Southern blot analysis. The analysis was conducted using wcor14a as a probe and total DNAs extracted from a diploid T. monococcum, tetraploids T. turgidum, T. durum and T. timopheevi, and common wheat cultivars M808 and CS. These DNAs were digested with four restriction enzymes (EcoRI, EcoRV, BamHI, or DraI) which did not cut at least inside the wcor14a cDNA and the genomic region studied. The result suggested that each of the constituent diploid genomes of the hexaploid wheat might possess at least two copies of wcor14-homologous sequences (Fig. 4). RFLP was also detected in the tetraploid and hexaploid wheat genomes studied. Cold-responsive gene expression of wcor14. We studied wcor14 transcript accumulation under the low temperature condition and other stress conditions. Total RNA extracted from M808 plants was analyzed by Northern blot hybridization using an entire cDNA of wcor14a as a probe. In the non-acclimated control plants, no wcor14 homologous transcripts were detected (Fig. 5A). Transcripts homologous to wcor14 rapidly accumulated within 3–6 hours after cold acclimation at 4°C (Fig. 5A lower panel). In most cases, a single intense signal and two additional weaker signals with higher molecular weights were detected. The amount of the major transcript reached a maximum at day 3 and thereafter leveled down but remained at a steady-state level until at least day 20 of the experiment (Fig. 5A upper panel). Turnover of the transcript also appeared to be rapid and it became non-detectable within 3 hours after transferring the plants back to the normal temperature condition (Fig. 5B upper panel). There were no differences in the transcript level during 3-days of cold acclimation between the standard light condition and the continuous darkness (Fig. 5B lower panel). Cold acclimation for additional 3 days under the continulous darkness did not affect the transcript level as compared to that observed in plants acclimated for 5 days under the standard light condition. Neither treatment with NaCl (400 mM) for 3 days, ABA (20 µM) for 3 days nor dehydration for 3 hours could induce detectable amounts of wcor14 transcript (Fig. 5C). The expression Fig. 3. Alignment of two intron sequences of wcor14 loci. Introns were amplified by the nested PCR using total DNA from M808 as a template and primer combinations of U1-L1 and U2-L2 (for the primer locations, see Fig. 1). Genomic sequences of two different sized introns (174 bp of intron I and 97 bp of intron II) were detected together with that without intron. A cold-responsive wheat gene wcor14 55 Fig. 4. Southern blot analysis of wcor14 in diploid, tetraploid and hexaploid wheat species. A: total DNAs were digested with EcoRI, BamHI or DraI and probed with the wcor14a cDNA. In each set, lane 1: T. turgidum (a genome constitution, AABB), lane 2: T. durum (AABB) and lane 3: T. timopheevi (AAGG). B: total DNAs were digested with EcoRV or DraI and probed with the wcor14a cDNA. In each set, lane 1: M808 (AABBDD), lane 2: CS (AABBDD), lane 3: T. durum (AABB) and lane 4: T. monococcum (AA). Fig. 5. Northern blot analysis of wcor14 transcripts. Total RNA was extracted from M808 plants. Plants were cold acclimated at 4°C under the standard 16L8D photoperiod for 0 to 20 days (upper panel A) or under continuous light for 0 to 24 hours (lower panel A). Upper panel B: plants were cold acclimated for 3 days under the standard photoperiod (3d0h) and then transferred back to the normal temperature condition for 3 hours (3d3h) and 12 hours (3d12h) under continuous light. Lower panel B: plants were cold acclimated under four combinations of the light conditions, for example 5 days under the standard photoperiod followed by 3 days under the continuous darkness (5d3d). Panel C: plants were treated with 400 mM NaCl or 20 µM ABA for 3 days (3d) or with dehydration for 3 hours (3h). All lanes were loaded with an equal amount of total RNAs in which no degradation was detected. 56 Sergei Tsvetanov et al. of wcor14 thus appeared to be low-temperature specific similar to the barley cor14b. Expression of the barley cor14b was reported to be strongly impaired in an albino mutant (Crosatti et al., 1999). Further, in etiolated barley plants cor14b gene expression and COR14b protein accumulation were both enhanced after exposure to a short light pulse with a high intensity of 200 µmol・m–2・s–1. These results demonstrated that the barley cor14b is cold-responsive and lightstimulated. The results also suggest the involvement of chloroplastic factor(s) in the control of its gene expression. By contrast, in our Northern blot analysis we found a similar abundance of the wcor14 major transcript during the cold acclimation under both continuous darkness for 3 days and the normal 16L8D light condition for 3 days (Fig. 5B lower panel). Acclimation for 5 days under the 16L8D condition followed by acclimation under continuous darkness for 3 days also did not affect the amount of wcor14 transcript. We applied illumination with a relatively low light intensity (55-65 µmol・m–2・sec–1) during the cold acclimation period. Moreover, the homoeologous wcor14 transcripts could not be distinguished individually by the Northern blot analysis. Our present results could at least suggest that light is not essential for the expression of all of the wcor14 homoeologous loci in the hexaploid wheat genome. Based on the present results, we conclude that wcor14 is a wheat orthologue of the barley cor14b. The expression of wheat wcor14 is low temperature specific. Further, its turnover appeared to be rapid and the expression remains at a high steady-state level during the cold acclimation period. A possible relation between the expression of wcor14 gene, accumulation of WCOR14 protein, and the freezing tolerance has to be further studied. The work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 10460006 to CN). We thank Dr. Cattivelli for kindly providing us with the cDNA clone bcor14b. The sequences of wcor14a and wcor14b cDNAs have been deposited in the GenBank data base (accession no. AF207545 and AF207546, respectively). Contribution no. 119 from the Laboratory of Plant Genetics, Kobe University. REFERENCES Cattivelli, L., and Bartel, D. (1990) Molecular cloning and characterization of cold- regulated genes in barley. Plant Physiol. 93, 1504 –1510. Chauvin, L.-P., Houde, M., and Sarhan, F. (1993) A leaf-specific gene stimulated by light during wheat acclimation to low temperature. Plant Mol. Biol. 23, 255 –265 . Chauvin, L.-P., Houde, M., and Sarhan, F. (1994) Nucleotide sequence of a new member of the freezing tolerance-associated protein family in wheat. Plant Physiol. 105, 1017–1018. Crosatti, C., Soncini, C., Stanca, A. M., and Cattivelli, L. (1995) The accumulation of a cold-regulated chloroplast protein is light-dependent. Planta 196, 458 –463. Crosatti, C., de Laureto, P. P., Bassi, R., and Cattivelli, L. (1999) The interaction between cold and light controls the expression of the cold-regulated barley gene cor14b and the accumulation of the corresponding protein. Plant Physiol. 119, 671– 680. Danyluk, J., Rassart, E., and Sarhan, F. (1991) Gene expression during cold and heat shock in wheat. Biochem. Cell Biol. 69, 383 –391. Danyluk, J., Houde, M., Rassart, E., and Sarhan, F. (1994) Differential expression of a gene encoding an acidic dehydrin in chilling sensitive and freezing tolerant Gramineae species. FEBS Lett. 344, 20 – 24. Danyluk, J., Carpentier, E., and Sarhan, F. (1996) Identification and characterization of a low temperature regulated gene encoding an actin-binding protein from wheat. FEBS Lett. 389, 324 –327. Gana, J. A., Sutton, F., and Kenefick, D. G. (1997) cDNA structure and expression patterns of a low temeprature-specific wheat gene tarc7. Plant Mol. Biol. 34, 643 –650. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97–120. Gavel, Y., and von Heijne, G. (1990) A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett. 261, 455 – 458. Graham, D., and Patterson, B. D. (1982) Responses of plants to low, non-freezing temperature: protein, metabolism, and acclimation. Annu. Rev. Plant Physiol. 33, 347– 372. Gray, G. R., Chauvin, L. P., Sarhan, F., and Huner, N. P. A. (1997) Cold acclimation and freezing tolerance. Plant Physiol. 114, 467– 474 Guo, W., Ward, R. W., and Thomashow, M. F. (1992) Characterization of a cold-regulated wheat gene related to Arabidopsis cor47. Plant Physiol. 100, 915 –922. Guy, C. L. (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 187– 223. Guy, C. L., Niemi, K. J., and Brambl, R. (1985) Altered gene expression during cold acclimation of spinach. Proc. Natl. Acad. Sci. U.S.A. 82, 3673 –3677. Houde, M., Danyluk, J., Laliberte, J. F., Rassart, E., Dhindsa, R. S., and Sarhan, F. (1992) Cloning, characterization, and expression of a cDNA encoding a 50- kilodalton protein specifically induced by cold acclimation in wheat. Plant Physiol. 99, 1381–1387. Houde, M., Daniel, C.,Lachapelle, M., Allard, F., Laliberte, S., and Sarhan, F. (1995) Immunolocalization of freezing-toleranceassociated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. Plant J. 8, 583 –593. Hughes, M. A., and Dunn, M. A. (1996) The molecular biology of plant acclimation to low temperature. J. Exp. Bot. 47, 291– 305. Jaglo-Ottosen, K. R., Glimour, S. J., Zaarka, D. G., Scgabenberger, O., and Thomashow, M. F. (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, 104 –106. Karlin-Neumann, G. A., and Tobin, E. M. (1986) Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework. EMBO J. 5, 9 –13 . Kurkela, S., and Franck, M. (1990) Cloning and characterization of a cold and ABA- inducible Arabidopsis gene. Plant Mol. Biol. 15, 137–144. Li, P. H., and Christersson, L. (1993) Advances in Plant Cold Hardiness. CRC Press, Boca Raton. A cold-responsive wheat gene wcor14 Lin, C., and Thomashow, M. F. (1992) DNA sequence analysis of a complementary DNA for cold-regulated Arabidopsis gene cor15 and characterization of the COR15 polypeptide. Plant Physiol. 99, 519 –525. Liu, Y. G., Mori, N., and Tsunewaki, K. (1990) Restriction fragment length polymorphism (RFLP) analysis in wheat. I. Genomic DNA library construction and RFLP analysis in common wheat. Jpn. J. Genet. 65, 367–380. Monroy, A. F., Castonguay, Y., Laberge, S., Sarhan, F., Vezina, L. P., and Dhindsa, R. S. (1993) A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature. Plant Physiol. 102, 873 –879. Neven, L. G., Haskell, D. W., Guy, C. L., Denslow, N., Klein, P. A., Green, J. G., and Silveman, A. (1992) Association of 70kilodalton heat-shock cognate proteins with acclimation to cold. Plant Physiol. 99, 1362 –1369. 57 Quellet, F., Houde, M., and Sarhan, F. (1993) Purification, characterization and cDNA cloning of the 200 kD protein induced by cold acclimation in wheat. Plant Cell Physiol. 34, 59 – 65. Thomashow, M. F. (1990) Molecular genetics of cold acclimation in higher plants. Adv. Genet. 28, 99 –131. Thomashow, M. F. (1994) Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. In: Arabidopsis (eds.: E. Meyerowitz, and C. Somerville), pp. 807–834. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Thomashow, M. F. (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol. 118, 1– 8. Veisz, O., and Stuka, J. (1990) Frost resistance studies with wheat in natural and artificial conditions. In: Proc. Int. Symp. on Cereal Adaptation to Low Temperature Stress (eds. I. Panayotov, and S. Pavlova), pp. 12 –17. Albena, Bulgaria.