Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H2O2 elevation
Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H2O2 elevation
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DOI：
10.1016/j.jplph.2011.08.002
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发表时间：
2012-01-01
影响因子：
4.3
通讯作者：
Lin, Yaw-Huei
中科院分区：
文献类型：
--
作者：
Chen, Hsien-Jung;Wu, Sin-Dai;Lin, Yaw-Huei
在本报告中，我们获得了一个全长的cDNA.SPCAT1是从乙烯利处理的甘薯L3成熟叶片中分离到的。SPCAT1的开放阅读框包含1479个核苷酸(492个氨基酸)，与拟南芥、茄子、灰红树、豌豆、马铃薯、烟草和番茄等几种植物过氧化氢酶具有较高的氨基酸序列同源性(约71.2-80.9%)。基因结构分析表明，SPCAT1编码一个过氧化氢酶，在其C末端含有一个保守的内部过氧化物体靶向信号PTS1基序和钙调蛋白结合域。RT-PCR结果表明，SPCAT1基因在成熟的L3和早衰的L4叶片中表达显著增强，而在未成熟的L1、L2和完全黄化衰老的L5叶片中显著降低。在暗处理和乙烯利处理的13片叶片中，SPCAT1的表达从0-24 h开始显著增强，然后逐渐下降，直到处理后72 h。SPCAT1基因的表达水平与过氧化氢的定性和定量含量也呈近似负相关。效应处理表明，抗氧化剂还原型谷胱甘肽、NADPH氧化酶抑制剂二苯基碘(DPI)、钙离子螯合剂EGTA和De-nova蛋白合成抑制剂放线菌酮均能抑制乙烯利诱导的SPCAT1表达。这些数据表明，活性氧的升高会产生过氧化氢。NADPH氧化酶、外源钙内流和从头合成的蛋白质是甘薯过氧化氢酶SPCAT1表达所必需的，并与乙烯利介导的表达增强有关。外源表达的过氧化氢酶SPCAT1融合蛋白延缓或缓解了乙烯利介导的叶片衰老和H_2O_2升高。根据这些数据，我们认为甘薯SPCAT1是一种乙烯利诱导的过氧化氢酶，其表达受还原型谷胱甘肽、DPI、EGTA和放线菌亚胺的调节。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过氧化氢动态平衡中发挥生理作用或功能。(C)2011年爱思唯尔股份有限公司。版权所有。
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 13 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24 h, then decreased gradually until 72 h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H2O2 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 nova protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H2O2. 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 H2O2 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 H2O2 homeostasis in leaves caused by developmental cues and environmental stimuli. (C) 2011 Elsevier GmbH. All rights reserved.