A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana*
A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana*
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DOI：
10.1074/jbc.m115.681601
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发表时间：
2015-12
期刊：
影响因子：
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通讯作者：
Yafei Qi;Xiayan Liu;Shuang Liang;Rui Wang;Yuanfeng Li;Jun Zhao;Jingxia Shao;Lijun An;Fei Y
中科院分区：
文献类型：
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作者：
Yafei Qi;Xiayan Liu;Shuang Liang;Rui Wang;Yuanfeng Li;Jun Zhao;Jingxia Shao;Lijun An;Fei Y
叶绿体是植物光合作用和许多其他重要代谢过程的场所，叶绿体发育是植物生长发育不可或缺的一部分。叶绿体发育缺陷的突变体可以表现出不同的颜色表型，包括耐人寻味的绿色表型，该表型在叶基表现为黄/白，在叶尖表现为绿色。通过大规模的遗传筛选，我们在拟南芥中发现了一系列新的绿色突变体，包括virescent3-1(vir3-1)、vir4-1和vir5-1。我们通过基于MAP的克隆，证明了VIR3编码一个可能的叶绿体金属蛋白酶。通过定点突变，我们证明了VIR3锌结合基序HeAGH中保守的组氨酸235残基是VIR3在叶绿体中积累所必需的。VIR3-GFP在叶片原生质体中的瞬时表达证实了VIR3在叶绿体中的定位。此外，利用表达VIR3-FLAG的转基因株系，我们证明了VIR3是一种内源性类囊体膜蛋白，主要存在于基质片层中。此外，利用表达双表位标记的VIR3的转基因株系进行的拓扑分析表明，VIR3的N端和C端都位于基质中，VIR3的催化结构域可能面向基质。蓝色天然凝胶分析表明，VIR3可能以单体或小复合体的形式存在于类囊体膜中。这项工作不仅暗示VIR3是参与叶绿体早期发育的一个新的因子，而且也为进一步了解叶绿体蛋白水解酶在叶绿体生物发生中的作用提供了更多的信息。
The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.