Department of Biological Sciences, National Sun Yat-sen University, 804 Kaohsiung, Taiwan.
J Plant Physiol. 2012 Jan 1;169(1):86-97. doi: 10.1016/j.jplph.2011.08.002. Epub 2011 Sep 4.
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.
在本报告中，从乙烯利处理的甘薯成熟 L3 叶片中分离出全长 cDNA SPCAT1。SPCAT1 的开放阅读框包含 1479 个核苷酸（492 个氨基酸），与几种植物过氧化氢酶（包括拟南芥、茄子、灰叶猴、豌豆、马铃薯、烟草和番茄）具有很高的氨基酸序列同一性（约 71.2-80.9%）。基因结构分析表明，SPCAT1 编码过氧化氢酶，其 C 末端含有一个假定的保守的内部过氧化物酶体靶向信号 PTS1 基序和钙调蛋白结合结构域。RT-PCR 显示，SPCAT1 基因在成熟的 L3 和早期衰老的 L4 叶片中表达显著增强，在不成熟的 L1、L2 和完全变黄衰老的 L5 叶片中表达显著降低。在黑暗和乙烯利处理的 L3 叶片中，SPCAT1 表达从 0 到 24h 暂时显著增强，然后逐渐下降，直到处理后 72h。SPCAT1 基因表达水平也与定性和定量 H2O2 量呈近似反比关系。效应物处理表明，抗氧化剂还原型谷胱甘肽、NADPH 氧化酶抑制剂二苯基碘鎓（DPI）、钙离子螯合剂 EGTA 和从头合成蛋白抑制剂环己酰亚胺抑制乙烯利增强的 SPCAT1 表达。这些数据表明，活性氧物种 H2O2、NADPH 氧化酶、外钙内流和新合成的蛋白质是必需的，并与乙烯利介导的甘薯过氧化氢酶 SPCAT1 表达增强相关。表达的过氧化氢酶 SPCAT1 融合蛋白的外源应用延迟或减轻了乙烯利介导的叶片衰老和 H2O2 升高。基于这些数据，我们得出结论，甘薯 SPCAT1 是一种乙烯利诱导的过氧化物酶，其表达受还原型谷胱甘肽、DPI、EGTA 和环己酰亚胺调节。甘薯过氧化氢酶 SPCAT1 可能在应对发育线索和环境刺激引起的叶片 H2O2 动态平衡中发挥生理作用或功能。
