ABSTRACT
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 genetic relationships using RNA sequencing (RNA-seq) and β-glucuronidase (GUS) staining. Using CRISPR/Cas9 technology, we constructed nine osckx mutants to determine the OsCKX function in rice development. Results revealed that each OsCKX gene has a unique expression pattern. Furthermore, the single osckx and higher-order osckx4 osckx9 mutant lines showed functional overlap and subfunctionalization. Mutant phenotypes associated with decreased CKX activity exhibited changes in leaf and root growth, inflorescence architecture, fertilization, and grain weight. 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 (WT). Moreover, we identified several genes that were significantly expressed in osckx4 and osckx9 single and double mutant plants. Many differentially expressed genes, such as OsPIN2, OsRR4, and OsNRT2.3, were found to be associated with auxin, cytokinin, and nitrogen pathways. Therefore, our findings provide new insights on the functions of OsCKX genes in rice growth, that may be used as a foundation for future studies aimed at improving rice yield and initiating green production.
One sentence summary Cytokinin oxidase/dehydrogenase family genes exhibit functional divergence and overlap in rice growth and development.
Footnotes
1 This work was supported by the National Natural Science Foundation of China (Grant number 31872855 and 31971842).
↵3 Senior author.
↵4 Author for contact: dingcq{at}njau.edu.cn.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Chengqiang Ding (dingcq{at}njau.edu.cn).
