Zhang Dan, Chang Enjie, Yu Xiaoxia, Chen Yonghuan, Yang Qinshuai, Cao Yanting, Li Xiukun, Wang Yuhua, Fu Aigen, Xu Min
Chinese Education Ministry's Key Laboratory of Western Resources and Modern Biotechnology, Key Laboratory of Biotechnology Shaanxi Province, College of Life Sciences, Northwest University, Xi'an, China.
Front Plant Sci. 2018 Jun 19;9:720. doi: 10.3389/fpls.2018.00720. eCollection 2018.
Soybean () seed yields rely on the efficiency of photosynthesis, which is poorly understood in soybean. Chlorophyll, the major light harvesting pigment, is crucial for chloroplast biogenesis and photosynthesis. Magnesium chelatase catalyzes the insertion of Mg into protoporphyrin IX in the first committed and key regulatory step of chlorophyll biosynthesis. It consists of three types of subunits, ChlI, ChlD, and ChlH. To gain a better knowledge of chlorophyll biosynthesis in soybean, we analyzed soybean Mg-chelatase subunits and their encoding genes. Soybean genome harbors 4 genes, 2 genes, and 3 genes, likely evolved from two rounds of gene duplication events. The qRT-PCR analysis revealed that , and genes predominantly expressed in photosynthetic tissues, but the expression levels among paralogs are different. In silicon promoter analyses revealed these genes harbor different -regulatory elements in their promoter regions, suggesting they could differentially respond to various environmental and developmental signals. Subcellular localization analyses illustrated that GmChlI, GmChlD, and GmChlH isoforms are all localized in chloroplast, consistent with their functions. Yeast two hybrid and bimolecular fluorescence complementation (BiFC) assays showed each isoform has a potential to be assembled into the Mg-chelatase holocomplex. We expressed each GmChlI, GmChlD, and GmChlH isoform in corresponding mutants, and results showed that 4 GmChlI and 2 GmChlD isoforms and GmChlH1 could rescue the severe phenotype of mutants, indicating that they maintain normal biochemical functions . However, GmChlH2 and GmChlH3 could not completely rescue the chlorotic phenotype of mutant, suggesting that the functions of these two proteins could be different from GmChlH1. Considering the differences shown on primary sequences, biochemical functions, and gene expression profiles, we conclude that the paralogs of each soybean Mg-chelatase subunit have diverged more or less during evolution. Soybean could have developed a complex regulatory mechanism to control chlorophyll content to adapt to different developmental and environmental situations.
大豆（）种子产量依赖于光合作用效率，而大豆光合作用效率目前还了解甚少。叶绿素是主要的光捕获色素，对叶绿体生物合成和光合作用至关重要。镁螯合酶在叶绿素生物合成的首个关键调控步骤中催化镁插入原卟啉IX。它由三种亚基组成，即ChlI、ChlD和ChlH。为了更好地了解大豆中的叶绿素生物合成，我们分析了大豆镁螯合酶亚基及其编码基因。大豆基因组含有4个基因、2个基因和3个基因，可能是由两轮基因复制事件进化而来。qRT-PCR分析显示，、和基因主要在光合组织中表达，但旁系同源基因之间的表达水平有所不同。在硅启动子分析中发现这些基因在其启动子区域含有不同的调控元件，表明它们可能对各种环境和发育信号有不同的反应。亚细胞定位分析表明，GmChlI、GmChlD和GmChlH异构体均定位于叶绿体，与其功能一致。酵母双杂交和双分子荧光互补（BiFC）分析表明，每个异构体都有可能组装成镁螯合酶全复合物。我们在相应突变体中表达了每个GmChlI以及GmChlD和GmChlH异构体，结果表明4个GmChlI和2个GmChlD异构体以及GmChlH1可以挽救突变体的严重表型，表明它们保持正常的生化功能，但GmChlH2和GmChlH3不能完全挽救突变体的黄化表型，这表明这两种蛋白质的功能可能与GmChlH1不同。考虑到在一级序列、生化功能和基因表达谱上显示的差异，我们得出结论，每个大豆镁螯合酶亚基的旁系同源基因在进化过程中或多或少已经发生了分化。大豆可能已经发展出一种复杂的调控机制来控制叶绿素含量，以适应不同的发育和环境情况。
