Fine mapping and functional analysis of the light-induced leaf curling gene <i>CaARF1</i> in pepper

Abstract This study investigated how light intensity and auxin regulate leaf curling in pepper using the natural curly leaf mutant D26 and the normal-leaf line A124. High light intensity significantly aggravated leaf curling in D26, and this effect was associated with compact arrangement of palisade and spongy mesophyll cells. Differential abaxial and adaxial application of exogenous auxin altered curling severity, indicating that polar auxin distribution is involved in this process. Genetic analysis confirmed that the curly leaf trait is controlled by a single recessive nuclear gene. By combining BSA-seq with KASP markers, the candidate interval was fine-mapped to a 1.5-Mb region on chromosome 09, leading to identification of CaARF1, which encodes an auxin response factor. CaARF1 localized to the nucleus. Virus-induced gene silencing of CaARF1 in pepper caused upward leaf curling, whereas its overexpression in tomato induced downward leaf twisting. Yeast two-hybrid, LCI, and BiFC assays demonstrated that CaARF1 specifically interacts with CaIAA4 (an Aux/IAA family member) in the nucleus. Silencing CaIAA4 partially rescued the curling phenotype of D26. Transcriptome analysis revealed that loss of CaARF1 function leads to hyperactivation of activating ARFs and abnormally high expression of cell-wall-relaxation genes such as XTH. Collectively, these findings show that CaARF1 interacts with CaIAA4 to integrate light signals and auxin responses, thereby maintaining cell wall remodeling homeostasis and regulating leaf flattening. This work provides a valuable genetic resource for leaf shape improvement and marker-assisted breeding in pepper.