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

Abstract

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.