Three novel subunits of Arabidopsis chloroplastic NAD(P)H dehydrogenase identified by bioinformatic and reverse genetic approaches.
Three novel subunits of Arabidopsis chloroplastic NAD(P)H dehydrogenase identified by bioinformatic and reverse genetic approaches.
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DOI：
10.1111/j.1365-313x.2008.03680.x
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
Atsushi Takabayashi;Noriko Ishikawa;T. Obayashi;S. Ishida;J. Obokata;T. Endo;F. Sato
中科院分区：
文献类型：
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作者：
Atsushi Takabayashi;Noriko Ishikawa;T. Obayashi;S. Ishida;J. Obokata;T. Endo;F. Sato
叶绿体NAD（P）H脱氢酶（NDH）在光系统I周围的循环电子流中发挥作用，产生ATP，特别是在适应环境变化方面。虽然NDH复合物含有11个与NADH：泛醌氧化还原酶（复合物I; EC 1.6.5.3）同源的亚基，但最近的遗传和生物学研究表明NDH也包含独特的亚基。我们在这里描述了一种基于共表达分析和系统发育分析的计算机模拟方法，用于识别65个基因作为NDH亚基的潜在候选基因。在这些ndh基因候选者中的21个拟南芥T-DNA插入突变体的表征表明，三个新的ndf（NDH依赖的循环电子流）突变体（ndf 1，ndf 2和ndf 4）有受损的NDH活性通过测量叶绿素荧光。在这些突变体中，NdhH亚基的量大大减少，这表明NDH活性的丧失是由NDH复合物积累缺陷引起的。此外，NDF 1、NDF 2和NDF 4蛋白与NdhH亚基共迁移，如蓝色天然电泳所示。这些结果有力地表明，NDF蛋白是NDH复合物的新亚基。进一步的分析表明，NDF 1和NDF 2蛋白在缺乏NDH复合物的疏水亚基的突变体中是不稳定的，但在缺乏亲水亚基的突变体中是稳定的，这表明NDF 1和NDF 2与疏水亚复合物相互作用。NDF 4蛋白具有氧化还原活性的铁硫簇结构域，可能参与电子传递。
Chloroplastic NAD(P)H dehydrogenase (NDH) plays a role in cyclic electron flow around photosystem I to produce ATP, especially in adaptation to environmental changes. Although the NDH complex contains 11 subunits that are homologous to NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), recent genetic and biological studies have indicated that NDH also comprises unique subunits. We describe here an in silico approach based on co-expression analysis and phylogenetic profiling that was used to identify 65 genes as potential candidates for NDH subunits. Characterization of 21 Arabidopsis T-DNA insertion mutants among these ndh gene candidates indicated that three novel ndf (NDH-dependent cyclic electron flow) mutants (ndf1, ndf2 and ndf4) had impaired NDH activity as determined by measurement of chlorophyll fluorescence. The amount of NdhH subunit was greatly decreased in these mutants, suggesting that the loss of NDH activity was caused by a defect in accumulation of the NDH complex. In addition, NDF1, NDF2 and NDF4 proteins co-migrated with the NdhH subunit, as shown by blue native electrophoresis. These results strongly suggest that NDF proteins are novel subunits of the NDH complex. Further analysis revealed that the NDF1 and NDF2 proteins were unstable in the mutants lacking hydrophobic subunits of the NDH complex, but were stable in mutants lacking the hydrophilic subunits, suggesting that NDF1 and NDF2 interact with a hydrophobic sub-complex. NDF4 protein was predicted to possess a redox-active iron-sulfur cluster domain that may be involved in the electron transfer.