miR156‐SPLs module regulates flowering and controls plant height by modulating gibberellin biosynthesis in citrus

Plant height and optimal flowering time are key determinants of crop yield and economic value. However, the regulatory mechanisms governing these traits, particularly in woody plants, remain unclear. In this study, overexpression of a citrus microRNA156 (miR156) family member, Ci-miR156c, resulted in significant phenotypic changes in citrus, including reduced plant height and delayed flowering. miR156-mediated repression of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) genes is a highly conserved regulatory mechanism in plants. Yeast one-hybrid and dual-luciferase assays, along with other related experiments, indicated that the Ci-miR156c-CiSPL7 module targets the bZIP transcription factor (CiFD) to regulate citrus flowering. Additionally, the Ci-miR156c-CiSPL6 module regulates plant height by targeting GA 20-oxidase 2 (CiGA20ox2), a key gibberellin biosynthesis gene. The Ci-miR156c-CiSPL3 module also influences plant height by regulating the KNOX family gene (CiKN6), which further regulates CiGA20ox2 expression. Overexpression of CiKN6 in citrus induced dwarfism, whereas its suppression increased height in transgenic plants, reinforcing its role in plant height regulation. Exogenous gibberellin and its inhibitor treatment further confirmed that the miR156-SPLs module regulates citrus plant height by inhibiting gibberellin biosynthesis. These findings highlight the role of the miR156-SPLs module in controlling citrus flowering and plant height.