The biosynthetic mechanism of a-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmaze) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of a-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.In an in vitro system using poly-(A) RNA isolated from rice scuteilum and the reticulocyte lysate translation system, a precursor form of aamylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of a-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of a-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of a-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of a-amylase were found to consist of three isozymes. The in vitro translated precursor forms of aamylase consisted of four multiple components. These results indicate that heterogeneity of a-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice a-amylase isozymes are encoded by multiple genes.In contrast to a long-held belief that a-amylase is synthesized de novo in the aleurone layers of germinating cereal seeds such as barley when triggered by gibberellic acid (4, 6, 12), recent experiments using histochemical techniques have demonstrated that the dominant site of formation ofa-amylase is the scutellar epithelium (24, 25). Electron microscopic observations clearly show the formation of membranous structures such as RER and Golgi apparatus in this tissue in early germination, suggesting the presence of a potentially active system of protein secretion in situ (26).In work reported in our previous publication (21), we demon-' Paper I I of the series is Reference 26 by Okamoto et al. (Plant Physiol, in press, 1982). This research was financially aided by the research grant from Mombusho (Japanese Ministry of Education) and the Mitsubishi Foundation (Tokyo).strated the in vitro synthesis of a-amylase directed by poly-(A) containing RNA isolated from the scutellar tissues in order to characterize the nature of a-amylase formation at the molecular level. It was found that the in vitro synthesized product had a lower mol wt than the mature secretory form of a-amylase molecule.During the biosynthesis of many secretory glycoproteins, an extrapeptide (signal peptide) recognizing the ER ...