Identification of Two Plastid-Targeted β-Amylases in Rice

In order to identify β-amylase isoforms acting inside plastids in rice (Oryza sativa L.), in the rice genome database we searched the genes predicted to encode β-amylase-like proteins, and designated them OsBAM1−9. OsBAM1, OsBAM7, and OsBAM9 genes were mainly expressed in germinating seeds and developing caryopses. In contrast, the transcripts of OsBAM2, OsBAM3, OsBAM4 and OsBAM5, which have a putative chloroplast transit peptide in their N-terminal regions, were mainly detected in leaf blades, leaf sheaths and internodes after heading. Soluble protein fractions prepared from Escherichia coli containing recombinant OsBAM2 and OsBAM3 proteins had significant β-amylase activity. Furthermore, OsBAM2-GFP and OsBAM3-GFP fusion proteins introduced into epidermis of Allium cepa scaly bulb were localized to the plastids. These results strongly suggest that OsBAM2 and OsBAM3 are plastid-targeted active β-amylase.