Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-Universität Munich, 82152 Planegg-Martinsried, Germany.
Plant Biochemistry, Faculty of Biology, Ludwig-Maximilians-Universität Munich, 82152 Planegg-Martinsried, Germany.
G3 (Bethesda). 2023 Sep 30;13(10). doi: 10.1093/g3journal/jkad187.
Plastids are the site of complex biochemical pathways, most prominently photosynthesis. The organelle evolved through endosymbiosis with a cyanobacterium, which is exemplified by the outer envelope membrane that harbors more than 40 proteins in Arabidopsis. Their evolutionary conservation indicates high significance for plant cell function. While a few proteins are well-studied as part of the protein translocon complex the majority of outer envelope protein functions is unclear. Gaining a deeper functional understanding has been complicated by the lack of observable loss-of-function mutant phenotypes, which is often rooted in functional genetic redundancy. Therefore, we designed outer envelope-specific artificial micro RNAs (oemiRs) capable of downregulating transcripts from several loci simultaneously. We successfully tested oemiR function by performing a proof-of-concept screen for pale and cold-sensitive mutants. An in-depth analysis of pale mutant alleles deficient in the translocon component TOC75 using proteomics provided new insights into putative compensatory import pathways. The cold stress screen not only recapitulated 3 previously known phenotypes of cold-sensitive mutants but also identified 4 mutants of additional oemiR outer envelope loci. Altogether our study revealed a role of the outer envelope to tolerate cold conditions and showcasts the power of the oemiR collection to research the significance of outer envelope proteins.
质体是复杂生化途径的所在地，其中最主要的是光合作用。这个细胞器通过与蓝细菌的内共生进化而来，这在外层膜上表现得尤为明显，质体的外层膜上有超过 40 种蛋白质，在拟南芥中就是如此。它们的进化保守性表明它们对植物细胞功能具有重要意义。虽然有少数几种蛋白质作为蛋白质易位复合物的一部分得到了很好的研究，但大多数外膜蛋白的功能仍不清楚。深入了解其功能变得复杂，因为缺乏可观察到的功能丧失突变体表型，这通常源于功能遗传冗余。因此，我们设计了质体外膜特异的人工 microRNA（oemiR），能够同时下调几个基因座的转录本。我们通过对 pale 和 cold 敏感突变体进行概念验证筛选来成功测试了 oemiR 的功能。利用蛋白质组学对缺乏易位成分 TOC75 的 pale 突变等位基因进行深入分析，为可能的补偿性导入途径提供了新的见解。冷应激筛选不仅重现了 3 种先前已知的冷敏感突变体表型，还鉴定了 4 种额外的 oemiR 外膜基因座的突变体。总的来说，我们的研究揭示了外膜在耐受低温条件方面的作用，并展示了 oemiR 集合在研究外膜蛋白重要性方面的威力。
