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• 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]

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG- Osh69 encoding alkaline alpha-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H2O2 and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal alpha-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards alpha-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence.

The structure and expression of a novel senescence-associated gene (SPA15) of sweet potato were characterized. The protein coding region of the gene consists of 13 exons encoding 420 amino acids. Apparent homologues of this sweet potato gene are found in a variety of dicot and monocot plants, but not in animals or microorganisms. Examination of the expression patterns of the SPA15 gene in sweet potato reveals that the transcripts of SPA15 are specifically induced in the senescing leaves, and the temporal profile of SPA15 protein accumulation is correlated with that of SPA15 transcripts. Studies on the distribution of SPA15 homologue in rice plants also indicate that SPA15 homologue is up-regulated specifically in senescing rice leaves. Treatment of detached sweet potato leaves with phytohormones including ethylene, methyl jasmonate, salicylic acid and abscisic acid resulted in a high-level induction of SPA15. Immunoelectron microscopic analysis demonstrates that SPA15 is specifically associated with the cell wall. The potential role for SPA15 during leaf senescence is discussed.