Comparative Studies for Deducing Functions of Chloroplast Structures in Green Plants

研究了绿单胞菌及其近缘种衣单胞菌的类pyrenoid形态和co2浓缩机制（CCM）的生理机制。根据所得结果，推测具有高浓度Rubisco分子的典型类芘的存在与CCM中大Ci池的形成有关。根据Rubisco大亚基基因rbcL的序列推断出这些衣单胞菌/绿单胞菌菌株与先前研究的无类pyrenoid绿单胞菌菌株之间的详细系统发育关系。两个单系类群得到了高自举值的解析。根据树的拓扑结构，我们推断典型类pyrenoid的丢失伴随着细胞内Ci池的减少可能在两组中独立发生。这是第一次证明叶绿体结构的具体功能。我们进一步比较了绿单胞菌与“绿单胞菌谱系”中近缘的含pyrenoid衣单胞菌的rbcL基因序列，以及与其他45种绿藻的rbcL基因序列。我们发现由rbcL基因编码的蛋白质在绿单胞菌谱系成员中比在其他藻类中具有更高水平的氨基酸替代。基于rbcL核苷酸序列的系统发育树将两个衣藻物种嵌套为单系衣藻“失pyrenoid”进化枝中的“重获pyrenoid”进化枝。然而，atpB和psaB序列数据都有力地支持了一组相当不同的系统发育关系，其中既没有解决“pyrenoid lost”的问题，也没有解决“pyrenoid lost”的问题。在基于rbcl的树中出现这样的分支可能是发生在大亚基区域的趋同进化变化的产物，该区域决定Rubisco分子是否会聚集形成可见的类pyrenoid。

The morphology of the pyrenoid and the physiology of the CO2-concentrating mechanism (CCM) were investigated in Chloromonas and related species of Chlamydomonas. Based on the results obtained, it was speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups. This is the first demonstration of concrete function of the chloroplast structure pyrenoid.We further compared sequences of the rbcL genes of pyrenoid-less Chloromonas species with those of closely related pyrenoid-containing Chlamydomonas species in the "Chloromonas lineage" and with those of 45 other green algae. We found that the proteins encoded by the rbcL genes had a much higher level of amino acid substitution in members of the Chloromonas lineage than they did in other algae. A phylogenetic tree based on rbcL nucleotide sequences nested two Chlamydomonas species as a "pyrenoid-regained" clade within a monophyletic Chloromonas "pyrenoid-lost" clade. However, both the atpB and the psaB sequence data gave robust support for a rather different set of phylogenetic relationships in which neither the "pyrenoid-lost" nor the "pyrenoid-regained" clade was resolved. The appearance of such clades in the rbcL-based tree may be an artifact of convergent evolutionary changes that have occurred in a region of the large subunit that determines whether Rubisco molecules will aggregate to form a visible pyrenoid.