Characterization of Arabidopsis mutants defective in the regulation of chlorophyllide a oxygenase
Characterization of Arabidopsis mutants defective in the regulation of chlorophyllide a oxygenase
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DOI：
10.1039/b802584n
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发表时间：
2008-10-01
影响因子：
3.1
通讯作者：
Tanaka, Ryouichi
中科院分区：
文献类型：
--
作者：
Kanematsu, Satoshi;Sakuraba, Yasuhito;Tanaka, Ryouichi
叶绿素B是陆地植物、绿色藻类和几种蓝藻产生的主要捕光色素之一。它是由叶绿素a通过叶绿素a加氧酶（chlorophyllide a oxygenase，CAO）合成的，在高等植物中CAO由三个结构域组成，即A、B。和C.我们以前证明，C结构域表现出催化功能，而A结构域不稳定的CAO蛋白在叶绿素B的存在下，从而调节细胞水平的CAO。在以前的研究中，我们也提出了遗传证据，证明参与的Clp蛋白酶在CAO的不稳定。本研究中，为了进一步了解CAO的调控机制，我们筛选了CAO积累控制缺陷的突变体。用甲基磺酸乙酯诱变过表达CAO A和B结构域与绿色荧光蛋白（GFP）嵌合蛋白的拟南芥转基因植物的种子。我们通过激光扫描共聚焦显微镜对转基因植株的后代进行筛选，共分离出66个表现出显著GFP荧光的突变体。通过免疫印迹分析，我们证实这些突变体在高水平上积累了CAO和GFP的N-末端结构域的融合蛋白。我们进一步将这些突变体分为七组的分布模式的融合蛋白，并通过色素和免疫印迹分析进行了表征。在此基础上，我们提出了一个模型来描述CAO的调控机制。
Chlorophyll b is one of the major light-harvesting pigments produced by land plants, green algae and several cyanobacterial species. It is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which in higher plants consists of three domains, namely, A, B. and C. We previously demonstrated that the C domain exhibits a catalytic function, whereas the A domain destabilizes the CAO protein in the presence of chlorophyll b, thus regulating the cellular level of CAO. In a previous study, we also presented genetic evidence demonstrating the involvement of Clp protease in the destabilization of CAO. In this study, in order to gain further insight into the regulatory mechanism of CAO, we screened for mutants defective ill the control of CAO accumulation. Seeds from an Arabidopsis transgenic plant overexpressing a chimeric protein consisting of the A and B domains of CAO and green fluorescent protein (GFP) were mutagenized by ethyl methane sulfonate. We screened the progenies of the transgenic plants by laser-scanning confocal microscopy, and isolated a total of 66 mutants exhibiting significant GFP fluorescence. By immunoblotting analysis, we confirmed that these mutants accumulated the fusion protein of the N-terminal domains of CAO and GFP at a high level. We further divided these mutants into seven groups by distribution patterns of the fusion protein, and characterized them by pigment and immunoblotting analyses. Based on these analyses, we proposed a model to describe the regulatory mechanism of CAO.