Cahoon A B, Timko M P
Department of Biology, University of Virginia, Charlottesville, Virginia 22903, USA.
Plant Cell. 2000 Apr;12(4):559-68. doi: 10.1105/tpc.12.4.559.
Light-independent protochlorophyllide reduction leading to chlorophyll formation in the dark requires both chloroplast and nuclear gene expression in Chlamydomonas reinhardtii. Mutations in any one of the plastid (chlL, chlN, and chlB) or nuclear (y-1 to y-10) genes required for this process result in the phenotype of the yellow-in-the-dark or y mutants. Analysis of the chlL, chlN, and chlB transcript levels in both light- and dark-grown wild-type and y mutant cells showed that the y mutations have no effect on the transcription of these plastid genes. Protein gel blot analysis showed that the CHLN and CHLB proteins are present in similar amounts in light- and dark-grown wild-type cells, whereas CHLL is present only in wild-type cells grown in the dark or at light intensities < or =15 micromol m(-2) sec(-1). Analysis of chlL transcript distribution on polysome profiles and rates of protein turnover in chloramphenicol-treated cells suggested that CHLL formation is most probably blocked at translation initiation or elongation. Furthermore, treatment of cells with metabolic inhibitors and uncouplers of photosynthetic electron transport showed that regulation of CHLL formation is linked to the physiologic status of the chloroplast. Similar to wild-type cells, y mutants contain nearly identical amounts of CHLN and CHLB when grown in either light or darkness. However, no CHLL is present in any of the y mutants except y-7, which contains an immunoreactive CHLL smaller than the expected size. Our findings indicate that CHLL translation is negatively photoregulated by the energy state or redox potential within the chloroplast in wild-type cells and that nuclear y genes are required for synthesis or accumulation of the CHLL protein.
在莱茵衣藻中，不依赖光的原叶绿素酸酯还原导致叶绿素在黑暗中形成，这需要叶绿体和细胞核基因表达。此过程所需的任何一个质体基因（chlL、chlN和chlB）或细胞核基因（y-1至y-10）发生突变，都会导致黑暗中发黄或y突变体表型。对野生型和y突变体细胞在光照和黑暗条件下生长时chlL、chlN和chlB转录水平的分析表明，y突变对这些质体基因的转录没有影响。蛋白质凝胶印迹分析表明，CHLN和CHLB蛋白在光照和黑暗条件下生长的野生型细胞中的含量相似，而CHLL仅存在于黑暗中或光照强度≤15 μmol m⁻² s⁻¹条件下生长的野生型细胞中。对chlL转录本在多核糖体图谱上的分布以及氯霉素处理细胞中蛋白质周转速率的分析表明，CHLL的形成很可能在翻译起始或延伸阶段受阻。此外，用光合电子传递的代谢抑制剂和解偶联剂处理细胞表明，CHLL形成的调控与叶绿体的生理状态相关。与野生型细胞类似，y突变体在光照或黑暗条件下生长时，CHLN和CHLB的含量几乎相同。然而，除了y-7之外，任何y突变体中都不存在CHLL，y-7含有一种免疫反应性CHLL，其大小小于预期大小。我们的研究结果表明，在野生型细胞中，CHLL翻译受到叶绿体能量状态或氧化还原电位的负向光调节，并且细胞核y基因是CHLL蛋白合成或积累所必需的。
