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

New Phytologist (2009)Summary; Here, we studied the functional role of a chloroplast alkaline a-galactosidase (OsAkaGal) in the breakdown of thylakoid membranes during rice (Oryza sativa) leaf senescence.; We assayed the enzyme activity of recombinant OsAkaGal with different natural substrates and examined the effect of ectopic OsAkaGal expression in rice plants.; Recombinant OsAkaGal 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 OsAkaGal null mutant (osakagal) displayed a delayed leaf senescence phenotype. OsAkaGal complementation in osakagal recovered OsAkaGal expression and showed a senescence phenotype similar to that of wild-type plants. Transgenic plants overexpressing OsAkaGal (UbiP-OsAkaGal) 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-OsAkaGal leaves also showed a 29-fold increase in alkaline a-galactosidase activity compared with wild-type leaves. An ultrastructural study of Ubi-OsAkaGal chloroplasts in newly unfolded leaves revealed abnormal grana organization.; Our findings strongly suggest that OsAkaGal is a thylakoid membrane-degrading enzyme involved in the degradation of digalactosyl diacylglycerol during rice leaf senescence.