The rice wx locus controls amylose synthesis in the endosperm. It contains two predominantly distributed functional alleles, Wx^a and Wx^b, which were initially defined on the basis of the amount of their gene products, Wx protein (Sano 1984). Wx^a allele produces about 10-fold higher levels of mRNA and protein than Wx^b does. Wx^b is present only in Japonica type of Oryza sativa, and Wx^a are widely distributed in Indica type of 0. sativa and various wild rice species including 0. rufipogon (Sano et al. 1991). Since Asian cultivated rice, Japonica and Indica, evolved from a common wild rice, 0. rufipogon, Wx^b is thought to have been derived from Wx^a. What kinds of mutations caused such a large change in the level of Wx gene expression during the relatively short period of the evolution of Japonica type? We have been studying gene expression of the Wx gene (Hirano amd Sano 1991) and we report here the molecular mechanism underlying the differences in the expression levels of Wx^a and Wx^b.

The rice Wx gene has a long intron (>l kb) at the upstream of ATG codon (Wang et al. 1995). DNA sequence (PI-DNA) that includes the promoter region (P) and the first intron (1) was fused to the GUS gene and GUS activity was analyzed in transient assay system using rice protoplasts. PI-DNA from Wx^a(from Indica cultivar Patpaku) and 0. rufipogon (W630) showed about 100-fold higher activity to drive GUS gene than that from Wx^b (from two Japonica strains Taichung 65 and Norm 8). To identify sequences responsible for a distinct difference in the expression level between two alleles, various chimeric sequences of PI-DNA consisting of Wx° (Indica) and Wx^b (Japonica, Taichung 65) were constructed and used for transient assay. The results indicated that the sequence responsible for the difference were not in the promoter region but in the downstream region (about 140 bp) of the transcriptional initiation site, where two sequence differences were detected between Wx^a (Indica) and Wx^b (Japonica). One was the splice donor site of the first intron. The donor sequence of Wx^b was TT, while that

Table 1. Comparisons of factors related to Wx alleles

Research Notes 149

of Wx^a was normal sequence, GT. Another difference was the number of TC repeats between the transcriptional initiation site and the First intron (Table 1). However, Wx^a derived from (9. rufipogon had the same sequence GT at the splice donor site and high GUS activity as well as Wx^a (Indica), while the number of TC repeats of Wx^a (0. ruflpogon) is more similar to that of Wx^b (Japonica) than Wx^a (Indica) (Table 1). These results indicated that the sequence difference responsible for the expression level between Wx^a and Wx^b is the change in the splice donor site of the first intron.

The northern blot analysis indicated Wx^a (Indica and 0. rufipogon) produces about 10-fold higher levels of its mRNA than does Wx^b. Moreover, the transcript from the seeds from Wx^b showed a larger band including the first intron in addition to the mature transcript, although no such band was detected in the transcript from all of Wx^a alleles. This indicates that the mutation at the splice donor site (GT to TT) in Wx^b reduced the efficiency of the splicing of the first intron. It seems likely that the low efficiency of splicing and accumulation of abnormal transcript in Japonica type causes the lower rate of transcription (by putative feed-back regulation) and/or that quick degradation of abnormal transcript results in a reduced level of the mature transcript.

Thus, we revealed that the distinct difference in the expression level between Wx^a and Wx^b resulted from the 1-bp change in the splice donor site. The expression level of the Wx gene affects the synthesis of amylose, whose content is the major determinant of rice quality, especially stickiness after cooking. Stickier rice, which is produced under the control of Wx^b allele, may have been selected during the domestication process of Japonica type. At molecular level, this selection corresponds to the screening of the 1 -base mutation, G to T, at the splice donor site of the first intron of the Wx gene. (Gene symbol: Old system)

Hirano,H.-Y.and Y.Sano, 1991. Molecular characterization of the waxy locus of rice (Oryza sativa). Plant

Sano, Y., 1984. Differential regulation of waxy gene expression in rice endosperm. Theor Appl Genet 68:

Sano, Y., H.-Y. Hirano and M. Nishimura, 1991. Evolutionary significance of differential regulation at the wx