The PtARF6/PtARF8-PtGH3.1 transcriptional module regulates auxin homeostasis and dwarfism in citrus

Dwarfing rootstocks are crucial for high-density and labor-efficient citrus cultivation, yet the molecular mechanisms underlying plant height regulation remain poorly understood. Using an integrated multi-omics analysis of dwarf and normal-height red tangerine (Citrus reticulata) × trifoliate orange (Poncirus trifoliata) hybrid seedlings, we identified the Gretchen Hagen 3.1 gene PtGH3.1, an auxin-conjugating enzyme, as an important regulator of dwarfism. Compared with normal-height hybrid seedlings, PtGH3.1 was significantly upregulated in the shoot tips of dwarf hybrid seedlings, correlating with reduced free auxin (indole-3-acetic acid, IAA) levels. Functional studies in a model citrus system (Citrus macrocarpa) demonstrated that heterologous overexpression of PtGH3.1 decreased free IAA and inhibited plant growth, whereas CRISPR/Cas9-mediated knockout of its homolog CmGH3.1 with editing ratio below 40% promoted growth. Mechanistically, the transcription factors auxin response factor 6 (PtARF6) and auxin response factor 8 (PtARF8) physically interact, forming a heterodimer that activates PtGH3.1 transcription. Dwarf hybrid seedlings exhibited elevated PtARF6 but reduced PtARF8 transcript levels. Heterologous overexpression of either PtARF6 or PtARF8 in Citrus macrocarpa induced dwarfism by activating CmGH3.1 expression. This study reveals a PtARF6/8-PtGH3.1 transcriptional module that regulates auxin homeostasis through IAA conjugation, providing genetic insights into GH3.1-mediated dwarfism and identifying precise targets (PtGH3.1, PtARF6, and PtARF8) for breeding dwarfing rootstocks in citrus.