Leaves are the primary vegetative organs of plants, and their morphology is an important trait affecting plant architecture, light energy utilization, environmental adaptation, and fruit quality and yield. Leaf development is highly flexible; however, understanding the regulatory mechanisms of factors coordinating leaf morphogenesis and differentiation remains limited. In this study, we obtained a double mutant for SlTCP29 and SlTCP24 genes from the CRISPR/Cas9 mutant population, both belonging to the CINCINNATA-like TCP (TEOSINTE BRANCHED, CYCLOIDEA and PCF1/2) transcription factor subfamily. Simultaneous mutations of SlTCP29 and SlTCP24 genes increase the complexity of tomato leaves, characterized by deeper leaf margin notches and increased number of leaflets. In conjunction with RNA-seq analysis, determination of plant hormone content, and molecular interaction assays, we identified the KNOXII gene SlTKNII5, SlMIR164a, and 1-aminocyclopropane-1-carboxylic acid synthase gene SlACS1A as direct downstream targets of SlTCP29 and SlTCP24, among which SlTKNII5 can physically interact with other KNOXII members to form heterodimers. Our study provides insight into the mechanisms by which SlTCP29 and SlTCP24 are involved in the morphological development of tomato compound leaves by integrating multiple pathways, including transcription factor, microRNA, and phytohormone.
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