Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1:: molecular biology, biochemistry and ultrastructure
Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1:: molecular biology, biochemistry and ultrastructure
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DOI：
10.1046/j.1365-313x.1999.00473.x
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发表时间：
1999-06-01
期刊：
影响因子：
7.2
通讯作者：
Herrmann, RG
中科院分区：
文献类型：
--
作者：
De Santis-Maciossek, G;Kofer, W;Herrmann, RG
peg介导的质体转化分别破坏了烟草质体编码RNA聚合酶亚基基因rpoA、B和C1。不同基因失活后产生的灰白色突变表型是相同的。突变体经历一个正常的个体发生周期，包括花的形成和可育种子的产生。它们的质体显示出发育不良的内部膜系统，由大的囊泡组成，偶尔有扁平的膜，使人想起堆积的类囊体。rpo（-）材料能够合成色素和脂质，成分相似，但含量低于野生型。西方分析表明，质体含有核编码的基质和类囊体多肽，包括最终处理的Sec管腔成分，但不包括Delta pH类囊体转运机制。使用后一种路径的组件作为中间体积累。与之形成鲜明对比的是，通过Western分析无法检测到由质体基因编码的参与光合作用的多肽，尽管如预期的那样发现了由核起源的质体RNA聚合酶转录的质体基因，包括rm操纵子。值得注意的是，超微结构、沉积和Northern分析以及脉冲实验表明，rpo（-）质体含有功能性核糖体。质体编码核糖体蛋白Rpl2的检测与这些结果一致。研究结果表明，rpo基因破坏的后果，以及两种质体聚合酶类型在整个细胞环境中的隐含整合，比目前假设的要复杂得多。
The plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo(-) material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the Delta pH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rm operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo(-) plastids contain functional ribosomes. The detection of the plastid-encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.