Abstract
The 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.