The α-amylase genes in Oryza sativa: Characterization of cDNA clones and mRNA expression during seed germination

Two cDNA clones, pOS103 and pOS137, were isolated which code for distinct α-amylase isozymes in germinating rice seeds. Sequence analysis indicated that the clones encode polypeptides of approximately 48 kDa, both of which possess a signal peptide involved in directing secretion of the protein. Comparison of the two rice α-amylase amino acid sequence showed that they are 76% similar to each other, while showing 85% to 90% similarity with other cereal α-amylases. A comparison of eleven cereal α-amylases also revealed three new conserved regions (I′, II′, and IV′) not previously identified in the animal, bacterial, and fungal α-amylases. Regions I′ and IV′ are sites for intron splicing while region II' is probably involved in calcium binding. One of the rice a-amylase cDNAs, pOS103, encodes a protein that has two potential N-glycosylation sites, one in the signal peptide and the other in the mature portion of the protein. The cDNA clone, pOS137, encodes an α-amylase with a single glycosylation site in the signal peptide, suggesting that the mature OS137 isozyme is not glycosylated. Analysis of the expression of these genes in germinating rice seeds indicated that mRNA corresponding to pOS103 and pOS137 could be detected throughout a 48 h period of seed imbibition. RNA levels, however, were dramatically stimulated by treatment of embryoless half-seeds with exogenous GA₃. Our results demonstrate that at least two forms of α-amylase are expressed in germinating rice seeds and that the expression of these genes is regulated by the phytohormone GA₃.