Cloning and characterization of a sweet potato calmodulin SPCAM that participates in ethephon-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression.

In this report a full-length cDNA, SPCAM, was isolated from ethephon-treated mature leaves of sweet potato. SPCAM contained 450 nucleotides (149 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 76-100%) with several plant calmodulins, including Arabidopsis, carrot, ghost needle weed, pea, potato, soybean, sweet chestnut, and tobacco. Sweet potato SPCAM also contained four putative conserved calmodulin EF-hand motifs, which responded for Ca(2+) binding and cellular signalling. Phylogenetic tree analysis showed that sweet potato SPCAM exhibited closely-related association with Arabidopsis AtCAM7, which functioned as a transcriptional regulator. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that SPCAM gene expression was not significantly increased from L1 immature leaf to L3 mature leaf, however, was remarkably enhanced in L4 early senescent leaf, and then decreased in L5 late senescent leaf. In dark- and ethephon-treated mature leaves, SPCAM expression was significantly increased from 6 to 48h, then decreased gradually until 72h after treatment. Ethephon-mediated leaf senescence, H(2)O(2) elevation, and senescence-associated gene expression, however, was remarkably inhibited by chlorpromazine, a calmodulin inhibitor. Exogenous application of purified calmodulin SPCAM fusion protein reversed the chlorpromazine repression of ethephon-mediated leaf senescence, H(2)O(2) elevation and senescence-associated gene expression. Based on these data we conclude that sweet potato SPCAM is an ethephon-inducible calmodulin and its expression is enhanced in natural and induced senescent leaves. Calmodulin SPCAM may play a physiological role in ethephon-mediated leaf senescence, H(2)O(2) elevation and senescence-associated gene expression in sweet potato leaves.

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H2O2 elevation.

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24h, then decreased gradually until 72h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H(2)O(2) amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H(2)O(2), NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H(2)O(2) elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H(2)O(2) homeostasis in leaves caused by developmental cues and environmental stimuli.