Characterization of senescence-associated gene expression and senescence-dependent and -independent cysteine proteases differing in microsomal processing in Anthurium

摘要

This study was conducted to identify senescence-regulated signaling pathways and genes in the monocot Anthurium andraeanum, L. for future efforts to modify leaf senescence via biotechnology. The Arabidopsis senescence-activated promoter (Pr-(SAG12)) fused to the gfp4 gene was expressed 20-fold higher, while the Pr35S-gfp4 was expressed 30% lower, in senescing relative to healthy leaves of Anthurium. This indicated that a senescence-activated pathway correctly regulated the Arabidopsis sag12 cysteine protease gene and suggested the presence of sag12 homologues in Anthurium. To identify sag12 homologs, two different Anthurium cysteine protease cDNAs, anth16 and anth17, were cloned and their sequences, expression, and protein products were characterized. The Anth17 and Anth16 protein sequences were 58 and 51% identical with Sag12, respectively. Of the two genes, anth17 expression most closely resembled the sag12 gene. Anth16 was expressed primarily in healthy immature leaves, while anth17 and a control gene (ubiquitin) were significantly induced during senescence of mature leaves. The levels of known internal standards, chlorophyll, mRNAs for cab (chlorophyll-a,b-binding protein) and psbA (D1 protein of PSII), decreased during senescence and served to verify the progression of senescence. Cytokinin treatments repressed anth17 mRNA accumulation and sustained psbA mRNA levels in senescing leaves. Sucrose moderately reduced anth17 and psbA mRNA levels. Anth16 has a unique secretory vesicle sorting motif in the C-terminus, and produces a 70kDa in vitro translation product that is post-translationally processed to 57 kDa by microsomal membranes, a property of secretory proteins. In contrast, anth17 was not processed and its intracellular retention is proposed to be necessary for senescence-dependent cellular disassembly and proteolysis