CRISPR‐Cas9‐mediated knockout of OsKCS11 in rice reveals potential crosstalk between very‐long‐chain fatty acids and cytokinin

SUMMARY Very‐long‐chain fatty acids (VLCFAs) play crucial roles in various physiological processes in plants. Through our investigation using a CRISPR‐Cas9 knockout mutant library in rice, we identified a semi‐dwarf rice mutant named CRISPR‐Cas‐based dwarf‐1 ( csd‐1 ). This mutant displayed multiple developmental defects, such as decreased plant height, panicle length, seed size, and seed‐setting rate. Whole‐genome resequencing analysis revealed that a T‐nucleotide insertion in β‐ketoacyl‐CoA synthase 11 (KCS11), responsible for the initial step in fatty acid elongation, was responsible for the observed defects in csd‐1 . The identity of csd‐1 was confirmed through genetic complementation and CRISPR‐Cas9‐mediated knockout. Expression analysis indicated that OsKCS11 was present in various tissues, with differential abundance observed through RT‐qPCR and promoter GUS staining, and strong localization at the node position by RNA in situ hybridization; furthermore, OsKCS11 protein was confirmed to be in the endoplasmic reticulum. Furthermore, csd‐1 exhibited significantly reduced levels of linolenic acid (18:3), C24:0‐OH, C28:0‐alkanes, C29:0‐alkanes, alpha‐tocopherol, and C33:0‐alkanes, while trans ‐nonadecenoic acid and behenic acid levels were increased. Cytokinin analysis revealed significant increases in isopentenyladenine (IPA) and cis ‐zeatin (cZ) levels in csd‐1 . Molecular investigations indicated upregulation of genes involved in cytokinin biosynthesis or signaling, suggesting a potential link between VLCFAs and cytokinin synthesis through acetyl‐CoA. This study not only proposed an alternative gene mapping method based on whole‐genome resequencing but also elucidated the mechanism by which VLCFAs influence cytokinin synthesis and signaling.