Yujie Zhou, Lifang Shi, Xia Li, Shaobo Wei, Xiangyuan Ye, Yuan Gao, Yupeng Zhou, Lin Cheng, Long Cheng, Fengying Duan, Mei Li, Hui Zhang, Qian Qian, Wenbin Zhou

Plant Biotechnology Journal；2024；IF：10.1

DOI：10.1111/pbi.14535

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosyntheticcarbon assimilation, as it catalyses the conversion  of inorganic carbon into organic carbon.Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substratespecificity. Improving the catalytic performance of RuBisCO is one of the key routes forenhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalyticproperties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understandingof functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPRCas9technology to randomly edit five rbcS genes in rice (OsrbcS1–5) , generating a series ofknockout mutants. The mutations of predominant rbcS genes in rice photosynthetic tissues, OsrbcS2–5 , conferred inhibited growth, delayed heading and reduced yield in the fieldconditions, accompanying with lower RuBisCO contents and activities and significantly reducedphotosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations.In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lowersugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of themutated RuBisCO enzyme in one rbcs2,3,5 mutant line uncovered no significant differencesfrom the wild-type protein, indicating that the mutations of rbcS did not compromise the proteinassembly or the structure. Our findings generated a mutant pool with genetic diversities, whichoffers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice.The multiplex genetic engineering approach of this study provides an effective and feasiblestrategy for RuBisCO modification in crops, further facilitate the photosynthesis improvementand sustainable crop production.