Abstract
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.
Highlights
From the $Research Institute for Biochemical Regulation,the **Department of Agricultural Ckmistv, School of Agriculture, and the //RadioisotopeCenter, Nagoya University, Chikusa,Nagoya 464-01, Japan
We have focused our research on phosphoenolpyruvatecarboxylase, pyruvate,orthophosphate dikinase, and the small subunit of ribulose-l,5-bisphosphatecarboxylase/oxygenase (RBUpzCase)which are coded by nuclear genes, and on the largesubunit of RBUpzCase which is coded by a plastid gene
There was an inverse correlationbetween the subsequent decarboxylation and refixation of this carbon by NADP-malic enzyme and the Ca photosynthetic carbon reduction cycle, respectively, in the bundle sheath (BS)’ cells [1].In fully green leaf tissue, the enzymes involved in the initial phases of the C, pathway, i.e. phosphoenolpyruvate carboxylase, pyruvate,orthophosphate dikinase, and NADPmalate dehydrogenase, are localized predominantly, if not exclusively, in the mesophyll cells, whereas NADP-malic enzyme, ribulose-1,5-bisphosphatecarboxylase/oxygenase (RBUpzCase),and several other CS cycleenzymes are located in the BS cells [1, 2]
Summary
Jarunya Ngernprasirtsiri$&Raymond CholletST, Hirokazu Kobayashill, Tatsuo Sugiyama**, and Takashi Akazawa$ $$. We have investigated the molecular regulatory mechanism(s)by which the nuclear and chloroplast genes encoding certain cell-specific C4 enzymes, e.g. phosphoenolpyruvate carboxylase, pyruvate,orthophosphate dikinase, and RBUp,Case are differentially expressed in the mesophyll and bundle sheath cells of a C4plant at different developmental stages, including etiolated, greening, and fully green leaves of maize. D N A Probes-For identifying specific genes and their transcripts, the following plasmids containing specific cloned DNA sequences were employed pZmc532 for maize plastid 16 S rDNA [32], pZmc461 for the large subunit (LS) of maize RBUpZCase (rbcL) 132),pSS15 for the small subunit (SS) of pea RBUp2Case( r b c S ) [33], pPPD1067 for maize pyruvate,orthophosphate dikinase ( p d k ) [34], and pM52 for maize phosphoenolpyruvate carboxylase ( p p c ) [35]. Based on analysis by light microscopy, chlorophyll a/b ratios, and the activitiesof cellspecific marker enzymes, the etiolated, greening, and fully green BS and MP preparations were adjudged to be free of
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