Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku-Katsura, Kyoto, Japan.
Center for Bioscience Research and Education, Utsunomiya University, Tochigi, Japan.
PLoS Biol. 2024 Sep 19;22(9):e3002785. doi: 10.1371/journal.pbio.3002785. eCollection 2024 Sep.
Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.
质体是通过质体工程全面增强植物光合作用和代谢特性的关键靶细胞器。质体蛋白主要来源于细胞核，必须穿过膜结合的多蛋白转运体才能进入这些细胞器。这个输入过程受到叶绿体靶向肽（cTPs）的精细调控。虽然已经有许多 cTP 被用于指导重组表达的功能性蛋白进入叶绿体，但仍迫切需要更有效的 cTP。在这里，我们对具有优越靶向能力的一系列先进 cTP 进行了全面探索和比较评估。我们采用了一种多方面的方法，包括计算预测、体内表达、荧光追踪和体外叶绿体导入研究，以鉴定和分析与拟南芥突变体相关的 88 个与叶绿体功能相关的 cTP。这些多肽具有将绿色荧光蛋白（GFP）运输到叶细胞内不同部位（尤其是叶绿体）的不同能力。来自拟南芥质体核糖体蛋白 L35（At2g24090）的高效 cTP 在叶绿体定位方面表现出显著的有效性。这种 cTP 促进了叶绿体靶向 RNA 加工蛋白和代谢酶在质体中的活性。这种 cTP 可以作为一种理想的转运肽，将生物分子精确靶向质体，从而推动质体工程的发展。
