Ngernprasirtsiri J, Chollet R, Kobayashi H, Sugiyama T, Akazawa T
Research Institute for Biochemical Regulation, Nagoya University, Japan.
J Biol Chem. 1989 May 15;264(14):8241-8.
The methylation of nuclear and chloroplast DNAs has been examined in relation to the known differential expression of C4 photosynthesis genes in the bundle sheath and mesophyll cells of etiolated, greening, and fully green maize leaves. We have focused our research on phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBUp2Case) which are coded by nuclear genes, and on the large subunit of RBUp2Case which is coded by a plastid gene. Reversed-phase high performance liquid chromatography revealed several kinds of methylated bases in DNAs of both photosynthetic cell types, with the largest amounts in fully green leaves. The occurrence of selective DNA methylation was investigated by employing an isoschizomeric pair of methyl-sensitive and -insensitive endonucleases followed by Southern hybridizations with specific DNA probes. Notably, there was an inverse correlation between the relative abundance of specific transcripts in a given cell type during greening and the methylation status of the corresponding nuclear or chloroplast gene. Furthermore, a heterologous in vitro transcription system using Escherichia coli RNA polymerase revealed that the plastid gene encoding the RBUp2Case large subunit in both cell types was active as a template in the unmethylated state, whereas it was inactive when methylated. Thus, the selective methylation of both chloroplast and nuclear DNA is likely one component of a multilevel control mechanism for the differential regulation of cell-specific C4 photosynthesis gene expression in greening maize leaves.
针对黄化、转绿和完全转绿的玉米叶片中，维管束鞘细胞和叶肉细胞已知的C4光合作用基因差异表达情况，研究了细胞核DNA和叶绿体DNA的甲基化。我们重点研究了由核基因编码的磷酸烯醇式丙酮酸羧化酶、丙酮酸磷酸双激酶和核酮糖-1,5-二磷酸羧化酶/加氧酶（RBUp2Case）小亚基，以及由质体基因编码的RBUp2Case大亚基。反相高效液相色谱法显示，两种光合细胞类型的DNA中均存在几种甲基化碱基，在完全转绿的叶片中含量最高。通过使用一对同裂酶（一种对甲基敏感，另一种对甲基不敏感）的内切核酸酶，随后用特异性DNA探针进行Southern杂交，研究了选择性DNA甲基化的发生情况。值得注意的是，在转绿过程中，特定细胞类型中特定转录本的相对丰度与相应核基因或叶绿体基因的甲基化状态呈负相关。此外，使用大肠杆菌RNA聚合酶的异源体外转录系统显示，两种细胞类型中编码RBUp2Case大亚基的质体基因在未甲基化状态下作为模板具有活性，而甲基化后则无活性。因此，叶绿体DNA和核DNA的选择性甲基化可能是玉米叶片转绿过程中细胞特异性C4光合作用基因表达差异调控的多级控制机制的一个组成部分。
