Optimizing cytokinin distribution patterns is a promising strategy for simultaneously enhancing grain yield, grain quality, and stress resistance in plants, as observed in ARGONAUTE2-overexpressing rice.

Brassinosteroids (BRs) regulate various agronomic traits such as plant height, leaf angle, and grain size in rice (Oryza sativa L.); thus BR signaling components are promising targets for molecular rational design. However, genetic materials for BR-signaling genes or family members remain limited in rice. Here, by genome editing using CRSPR/Cas9 tools, we generated a panel of single, double, triple or quadruple mutants within three BR signaling gene families, including GSK3/SHAGGY-LIKE KINASE1 (GSK1)-GSK4, BRASSINAZOLE-RESISTANT1 (OsBZR1)-OsBZR4, and PPKL1-PPKL3 (encoding protein phosphatases with kelch-like repeat domain), under the same background (Zhonghua11, japonica). The high-order mutants were produced by either simultaneously targeting multiple sites on different genes of one family (GSKs and PPKLs) or targeting the overlapping sequences of family members (OsBZRs). The mutants exhibited a diversity of plant height, leaf angle, and grain morphology. Comparison analysis of the phenotypes together with BR sensitivity tests suggested the existence of functional redundancy, differentiation, or dominancy among the members within each family. In addition, we generated a set of transgenic plants overexpressing GSK2, OsBZR1/2, and PPKL2, respectively, in wild-type or activated forms with fusion of different tags, and also verified the protein response to BR application. Collectively, these plants greatly enriched the diversity of important agronomic traits in rice. We propose that editing of BR-related family genes could be a feasible approach for screening of desired plants to meet different requirements. Release of these materials as well as the related information also provides valuable resources for further BR research and utilization.

No abstract