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

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