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. [ABSTRACT FROM AUTHOR]