Low temperature induces expression of the Cor/Ltr gene family in wheat. Wcor14 and Wcor15 genes are responsive to cold and light, and they contain the same signal peptide for targeting into chloroplast. Their expression patterns such as cold-responsiveness and cultivar differences are also similar. These observations indicate that the 5' upstream sequences of these two Cor genes contain the same cis-regulatory elements. To analyze the 5' regulatory sequences, we isolated a Wcor15 genomic clone from the wheat genomic library and determined its nucleotide sequences. The 5' upstream region of Wcor15 contains four CRT/DRE motifs, which are known as responsive motifs to low temperature and light. The 5' upstream sequence of the Wcor15 was fused to GUS reporter gene and introduced into tobacco genome. Both low temperature and light induced the GUS expression in the transgenic tobacco leaves. The expression pattern of the GUS gene in the tobacco leaves was well consistent with that of the Wcor15 gene in the native wheat seedlings. To characterize WCOR15 protein, the Wcor15 gene was expressed in E. coli. After centrifugation, the extracted histidine-tagged WCOR15 protein remained in the insoluble fraction. However, after solubilized with urea, the purified protein showed boiling- soluble feature. These results suggest that the WCOR15 protein interacts with membranes and plays a chaperon-like role to protect the membrane integrity against cold injury in the wheat chloroplasts.

P-6 R. Morimoto¹, Y. Ogihara², C. Nakamura¹ and S. Takumi¹ (¹Fac Agr, Kobe Univ, ² Kihara Inst Biol Res, Yokohama City Univ)
Mutations accumulated in the wheat Kn1 -type homeobox gene Wknox1

The plant knotted 1 (kn1)-like homeobox (knox) genes are known to play important roles in the maintenance of shoot apical meristem (SAM), determination of cell fate and differentiation of vegetative tissues. Three homoeologous cDNAs encoding the kn1-like homeobox protein were previously isolated and designated as Wknox1a, Wknox1b and Wknox1d. These were assigned to the homoeologous group 4 chromosomes. To investigate accumulation of mutations in the three homoeologous loci of wheat genome, we compared three homoeologous genomic sequences of the Wknox1 region. The three Wknox1 genes consisted of six exons and five introns. In the exon/intron regions, 29 mutations of insertion/deletion (more than 6bp in length) were found and most of them were located in the 4th intron. Four insertions, which were found in the 4th intron of Wknox1a, Wknox1b and Wknox1d, belonged to the Stowaway family of MITEs. The insertion events might have occurred in various times during wheat evolution, such as diversification of the ancestral diploid species, amphidiploidization and domestication of tetraploid wheat. The mutation rate in the Wknox1b locus was higher than that in the Wknox1a and Wknox1d loci. Moreover, the same expression level was observed of the three Wknox1 loci by RT-PCR analysis, thus no mutations in the three homoeologous Wknox1 loci affected the transcription level. The Wknox1a locus therefore can not be considered as a candidate locus for the Hooded phenotype in wheat.

P-7 K. Mizumoto¹, K. Murai², C. Nakamura¹ and S. Takumi¹ (¹Fac Agr, Kobe Univ, ² Fukui Pref Univ)
Cloning of genes involved in wheat ovule development and their expression in pistillate stamens

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in the alloplasmic line of common wheat with Aegilops crassa cytoplasm. The induction of pistillody is suppressed by Rfd1 gene located on the long arm of chromosome 7B in Chinese Spring (CS). Because of the absence of Rfd1, the alloplasmic line of CS ditelosomic 7BS((cr)-CSdt7BS) exhibits pistillody in all florets, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) with normal cytoplasm forms normal stamens. (cr)-CSdt7BS exhibit not only pistillody but also female sterility. In the (cr)-CSdt7BS pistils, abnormal ovules fail to form inner epidermis and integuments in the chalazal region. The pistillate stamens also show incomplete ovule-like structures either with inner integuments or with both inner and outer integuments. To study ovule development of wheat and the effect of Ae. crassa cytoplasm on the ovule formation, we cloned a wheat BEL1 homologue Wbel1 and an ANT(AINTEGUMENA) homologue Want. Both BEL1 and ANT are essential for normal ovule development in Arabidopsis thaliana. According to RT-PCR analysis of the isolated genes, we detected the Want transcript specifically expressed in pistils and pistillate stamens of both CSdt7BS and (cr)-CSdt7BS. However, the expression level of Wbel1 in the pistils and pistillate stamens was highly reduced in both (cr)-CS and (cr)-CSdt7BS. These results suggest that the expression of Wbel1 and Want is related to the pistillody phenotype and that the Ae. crassa cytoplasm affects the Wbel1 expression level.

P-8 K. Uno¹, D. Ogawa¹, H. Tsujimoto², K. Noda³ and N. Kawakami¹ (¹Fac Agric, Meiji Univ, ²Fac Agric, Tottori Univ, ³Res Inst Biol Res, Okayama Univ)
Analysis of seed dormancy and phytohormone crosstalk in Rht-B1c near isogenic line

Abscisic acid (ABA) and gibberrelic acid (GA) have been proved to be involved in grain dormancy and germination. However, the contribution of GA and its signaling on dormancy has been still obscure. We used a near isogenic line of wheat, Rht-B1c, which was GA insensitive in genetic background of CS, to see the effect of GA sensitivity on grain dormancy. At grain maturity, only 3 to 7% of Rht-B1c grains germinated, whereas more than 90% of CS grains germinated after imbibition at 22^oC for 7 days. Germinability of Rht-B1c increased along with time of storage at room temperature (after-ripening), and almost all the grains germinated after 30 to 40 days. This clearly suggests that Rht-B1c allele invests the seeds with dormancy. Half grains with embryos of Rht-B1c showed higher sensitivity to ABA than CS during the after-ripening. Rht-B1c allele might affect on dormancy development and/or maintenance by enhancing ABA signaling via its GA insensitive character. We have amplified fragments of Rht gene, and identification of the sequence of Rht-B1c allele is now in progress. We are trying to clone and identify downstream genes, which expressions are affected by Rht, with PCR-aided subtraction method and microarray analysis.