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 gene Osl2, which is specifically upregulated during leaf senescence in rice (Oryza sativa L. cv. Tainong 67), was cloned and functionally characterized. The protein coding region of the gene consists of 19 exons encoding 516 amino acids, with a putative mitochondrial targeting sequence and conserved sequence of the pyridoxal 5 0 -phosphate-binding domain. The recombinant Osl2 fusion protein over-expressed in Escherichia coli displays pyruvate-dependent g-aminobuty-ric acid (GABA) transaminase (EC 2.6.1.19) activity. Examination of the expression patterns of the Osl2 gene in rice reveals that Osl2-specific transcripts are induced in the senescing leaves. The temporal profile of Osl2 protein accumulation is correlated with that of pyruvate-dependent GABA transaminase activity in rice leaves, with the highest expression level at the S3 senescent stage. The potential role for GABA transaminase during rice leaf senescence is discussed.

Summary• The cellular events associated with programmed cell death during leaf senescence in rice (Oryza sativa) plants are reported here.• The cytological sequence of senescence-related changes in rice leaves was studied by transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DNA ladder assay.• Cell death in senescing mesophyll cells was marked by depletion of cytoplasm in a tightly controlled manner. However, no apparent morphological feature associated with apoptosis was observed. Nuclear DNA fragmentation was detectable as early as during leaf unfolding and the subsequent developmental and senescent stages. The occurrence of DNA fragmentation correlated well with the size-shift of chromosomal DNA on agarose gel after electrophoresis. However, DNA fragmentation was not accompanied by generation of oligonucleosomal DNA fragments.• These features of cell death occurring during leaf senescence in monocot rice are quite different from features characteristic of apoptosis in animals. The implications of these results for cellular events associated with rice leaf senescence are discussed.