State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 100081, People's Republic of China.
Plant Mol Biol. 2016 Nov;92(4-5):581-595. doi: 10.1007/s11103-016-0533-0. Epub 2016 Aug 29.
Plastid-encoded plastid RNA polymerase (PEP), a dominant RNA polymerase in mature chloroplasts, consists of core subunits and peripheral subunits. Despite the importance of the peripheral subunits in control of PEP activity it is unclear how they interact with one another to exert physiological effects on chloroplast development and plant growth, especially in rice. Here, we report a mutant, designated wsl3 that lacks a peripheral subunit in rice. We isolated the WSL3 gene encoding an essential peripheral subunit of rice PEP complex, OsPAP1/OspTAC3 by map-based cloning, and verified its function by complementation analysis. The wsl3 mutant showed a typical expression pattern of plastid-encoded genes, suggesting that PEP activity was impaired. Using immunofluorescent labeling and immunoblotting, we found that WSL3 was localized to the chloroplast and associated with the nucleoid. In addition, we demonstrated that WSL3 interacted with PEP subunits in Y2H, BiFC and pull-down experiments. Furthermore, a cpDNA IP assay revealed that WSL3 was associated with the PEP complex during the entire transcription process. We provide evidence suggesting that WSL3 is essential for early chloroplast development by interacting with subunits of the PEP complex.
质体编码的质体 RNA 聚合酶(PEP)是成熟叶绿体中主要的 RNA 聚合酶,由核心亚基和外周亚基组成。尽管外周亚基在控制 PEP 活性方面非常重要,但目前尚不清楚它们如何相互作用,以对叶绿体发育和植物生长产生生理影响,尤其是在水稻中。在这里,我们报道了一个水稻 wsl3 突变体,该突变体缺乏 PEP 复合物的外周亚基。我们通过图谱克隆分离了编码水稻 PEP 复合物必需外周亚基的 WSL3 基因 OsPAP1/OspTAC3,并通过互补分析验证了其功能。wsl3 突变体表现出典型的质体编码基因表达模式,表明 PEP 活性受损。通过免疫荧光标记和免疫印迹,我们发现 WSL3 定位于叶绿体,并与类核结合。此外,我们在 Y2H、BiFC 和 pull-down 实验中证明了 WSL3 与 PEP 亚基相互作用。此外,cpDNA IP 测定表明,WSL3 在整个转录过程中都与 PEP 复合物相关联。我们提供的证据表明,WSL3 通过与 PEP 复合物的亚基相互作用,对于早期叶绿体发育是必需的。