Alkaline α-galactosidase degrades thylakoid membranes in the chloroplast during leaf senescence in rice

P> Here, we studied the functional role of a chloroplast alkaline alpha-galactosidase (OsAk alpha Gal) in the breakdown of thylakoid membranes during rice (Oryza sativa) leaf senescence. We assayed the enzyme activity of recombinant OsAk alpha Gal with different natural substrates and examined the effect of ectopic OsAk alpha Gal expression in rice plants. Recombinant OsAk alpha Gal showed at least a two-fold greater substrate-binding affinity and a 10-fold greater turnover rate to galactolipid digalactosyl diacylglycerol than the raffinose family of oligosaccharides (verbascose, stachyose, raffinose) and melibiose. The OsAk alpha Gal null mutant (osak alpha gal) displayed a delayed leaf senescence phenotype. OsAk alpha Gal complementation in osak alpha gal recovered OsAk alpha Gal expression and showed a senescence phenotype similar to that of wild-type plants. Transgenic plants overexpressing OsAk alpha Gal (UbiP-OsAk alpha Gal) exhibited retarded plant growth and development, and showed a pale-green phenotype coupled with a reduced chlorophyll content to 42% in newly unfolded leaves. UbiP-OsAk alpha Gal leaves also showed a 29-fold increase in alkaline alpha-galactosidase activity compared with wild-type leaves. An ultrastructural study of Ubi-OsAk alpha Gal chloroplasts in newly unfolded leaves revealed abnormal grana organization. Our findings strongly suggest that OsAk alpha Gal is a thylakoid membrane-degrading enzyme involved in the degradation of digalactosyl diacylglycerol during rice leaf senescence.