Alkaline alpha-galactosidase degrades thylakoid membranes in t...

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