Center for Gene Research, Nagoya University, Nagoya, 464-8602, Japan.
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, 464-8602, Japan.
Plant J. 2019 Mar;97(6):1120-1131. doi: 10.1111/tpj.14187. Epub 2019 Jan 18.
Chloroplast gene expression is controlled by numerous nuclear-encoded RNA-binding proteins. Among these, pentatricopeptide repeat (PPR) proteins are known to be key players in post-transcriptional regulation in chloroplasts. However, the functions of many PPR proteins remain unknown. In this study, we characterized the function of a chloroplast-localized P-class PPR protein PpPPR_21 in Physcomitrella patens. Knockout (KO) mutants of PpPPR_21 exhibited reduced protonemata growth and lower photosynthetic activity. Immunoblot analysis and blue-native gel analysis showed a remarkable reduction of the photosystem II (PSII) reaction center protein and poor formation of the PSII supercomplexes in the KO mutants. To assess whether PpPPR_21 is involved in chloroplast gene expression, chloroplast genome-wide microarray analysis and Northern blot hybridization were performed. These analyses indicated that the psbI-ycf12 transcript encoding the low molecular weight subunits of PSII did not accumulate in the KO mutants while other psb transcripts accumulated at similar levels in wild-type and KO mutants. A complemented PpPPR_21KO moss transformed with the cognate full-length PpPPR_21cDNA rescued the level of accumulation of psbI-ycf12 transcript. RNA-binding experiments showed that the recombinant PpPPR_21 bound efficiently to the 5' untranslated and translated regions of psbImRNA. The present study suggests that PpPPR_21 may be essential for the accumulation of a stable psbI-ycf12mRNA.
质体基因表达受许多核编码 RNA 结合蛋白调控。其中，五肽重复（PPR）蛋白被认为是质体中转录后调控的关键因子。然而，许多 PPR 蛋白的功能仍然未知。在这项研究中，我们对拟南芥质体定位的 P 类 PPR 蛋白 PpPPR_21 的功能进行了研究。PpPPR_21 的敲除（KO）突变体表现出生长缓慢和光合活性降低。免疫印迹分析和蓝色非变性凝胶分析表明，KO 突变体中的光系统 II（PSII）反应中心蛋白显著减少，PSII 超复合物形成不良。为了评估 PpPPR_21 是否参与质体基因表达，进行了质体全基因组微阵列分析和 Northern blot 杂交。这些分析表明，编码 PSII 低分子量亚基的 psbI-ycf12 转录本在 KO 突变体中没有积累，而其他 psb 转录本在野生型和 KO 突变体中积累水平相似。用编码全长 PpPPR_21cDNA 的互补 PpPPR_21KO 苔藓转化体挽救了 psbI-ycf12 转录本的积累水平。RNA 结合实验表明，重组 PpPPR_21 能够有效地结合 psbImRNA 的 5'非翻译区和翻译区。本研究表明，PpPPR_21 可能对 psbI-ycf12mRNA 的稳定积累至关重要。
