A novel alkaline alpha-galactosidase gene is involved in rice leaf senescence

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG- Osh69 encoding alkaline alpha-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H2O2 and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal alpha-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards alpha-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence.