SummaryThe chloroplast genome has considerable potential to enhance crop productivity, but it remains underutilized in breeding because it is difficult to modify. This study elucidates the potential of recently developed chloroplast‐targeted C‐to‐T base editors in facilitating the use of the chloroplast genome for crop breeding. The herbicide metribuzin interferes with photosynthesis by binding to the D1 protein of photosystem II, encoded by the chloroplast genome. Naturally occurring D1 mutants with V219I or A251V substitutions are known to have resistance to some herbicides including metribuzin. Here, using the base editors, we introduced these substitutions and showed that the A251V single mutation and the V219 & A251V double mutations conferred significant metribuzin resistance to Arabidopsis thaliana. The V219I & A251V double mutants exhibited increased metribuzin resistance and grew better than the A251V single mutants. Furthermore, the double mutants grew as well as wild‐type plants in the absence of metribuzin. The single and double mutants, which are a challenge to obtain through traditional mutagenesis and crossbreeding methods, can be relatively easily generated using C‐to‐T base editors. In view of the conservation of V219 and A251 across numerous species, C‐to‐T base editing can potentially confer metribuzin resistance to a wide range of crops. Compared to nuclear genes, chloroplast genes are also less likely to spread into wild populations. Our findings suggest that chloroplast‐targeting C‐to‐T base editors will find many roles in future crop breeding efforts.

中文翻译：

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通过叶绿体基因组的靶向碱基编辑赋予拟南芥的除草剂 metribuzin 的抗性


摘要叶绿体基因组在提高作物生产力方面具有相当大的潜力，但由于难以修饰，它在育种中仍未得到充分利用。本研究阐明了最近开发的叶绿体靶向 C-to-T 碱基编辑器在促进叶绿体基因组用于作物育种方面的潜力。除草剂 metribuzin 通过与叶绿体基因组编码的光系统 II 的 D1 蛋白结合来干扰光合作用。已知具有 V219I 或 A251V 取代的天然 D1 突变体对某些除草剂（包括 metribuzin）具有抗性。在这里，使用碱基编辑器，我们介绍了这些替换，并展示了A251V单突变和V219和A251V双突变赋予了对拟南芥的显著metribuzin抗性。V219I & A251V双突变体表现出更高的metribuzin抗性，并且比A251V单突变体生长得更好。此外，在没有 metribuzin 的情况下，双突变体和野生型植物一样生长。通过传统的诱变和杂交方法获得单突变体和双突变体是一个挑战，使用 C-to-T 碱基编辑器可以相对容易地生成。鉴于 V219 和 A251 在许多物种中的保守性，C-to-T 碱基编辑可能会赋予 metribuzin 对多种作物的抗性。与核基因相比，叶绿体基因也不太可能扩散到野生种群中。我们的研究结果表明，靶向叶绿体的 C-to-T 碱基编辑器将在未来的作物育种工作中发挥许多作用。