Cytokinin oxidase/dehydrogenase family genes exhibit functional divergence and overlap in rice growth and development

Cytokinin has important functions during plant growth and development; hence, many researchers have extensively studied cytokinin biosynthesis and degradation. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis. In rice, 11 OsCKX genes have been identified to date; however, most of their functions remain unknown. Here, we comprehensively analyzed the expression patterns of OsCKX genes and their functions. Using CRISPR/Cas9 technology, we constructed nine osckx mutants to determine the OsCKX function in rice development. The results revealed that the single osckx and higher-order osckx4 osckx9 mutant lines showed functional overlap and subfunctionalization. Notably, we found that the osckx1 osckx2 and osckx4 osckx9 double mutants displayed contrasting phenotypic changes in tiller number, culm diameter, and panicle size as compared to the wild-type. Moreover, we identified several genes that were significantly expressed in osckx4 and osckx9 single and double mutant plants. Many differentially expressed genes were found to be associated with auxin, cytokinin, and nitrogen pathways. Therefore, our findings provide new insights into the functions of OsCKX genes in rice growth, which may be used as a foundation for future, studies aimed at improving rice yield and initiating green production.