AFL transcription factors play crucial roles for ricinoleic acid and storage lipid biosynthesis in castor bean seeds

Abstract

AFL transcription factors (ABI3, FUS3, and LEC2) are key regulators of seed development and lipid metabolism in plants; however, their regulatory network and target genes remain poorly characterized in castor bean (Ricinus communis), an important oil crop. This study aimed to elucidate the regulatory role of RcAFL transcription factors in triacylglycerol (TAG) and hydroxy fatty acid biosynthesis in castor bean seeds. Using DNA affinity purification sequencing (DAP-seq), we detected the whole-genome binding sites of RcABI3, RcFUS3, and RcLEC2, identifying 775 potential target genes including 62 lipid-related genes. Electrophoretic mobility shift assays (EMSA) revealed distinct cis-regulatory element preferences: RcFUS3 bound canonical RY motifs (5′-CATGCA-3′), RcLEC2 recognized RY-like motifs (5′-CATGNNCATGCA-3′), and RcABI3 targeted a novel AGCAATGA motif. Yeast one-hybrid and luciferase reporter assays validated direct binding and transcriptional activation of key lipid-related genes by RcFUS3, including a gene encoding FAH12 critical for ricinoleic acid synthesis. Furthermore, RcFUS3 directly activated the expression of RcWRI1, a master regulator of oil biosynthesis, establishing a hierarchical regulatory loop that amplifies the lipid biosynthesis pathway. Importantly, transient overexpression of RcFUS3 in castor bean cotyledon protoplasts significantly increased TAG content, and ChIP-qPCR confirmed that RcFUS3 directly binds to the RY motifs in the promoters of Oleosin2, PDAT2, and RcWRI1 in vivo. These findings demonstrate that RcAFL transcription factors coordinate TAG biosynthesis in castor bean seeds by binding distinct cis-elements, with RcFUS3 serving as a key regulator of lipid-related genes, providing valuable information toward understanding the potential regulatory network of AFL in castor beans.