PagARF3.1 promotes adventitious root formation by repressing <i>IPT</i>-mediated cytokinin biosynthesis

Adventitious root formation is a crucial developmental process in the clonal propagation of economically significant horticultural and woody species. This process is antagonistically regulated by auxin and cytokinin, yet the underlying molecular regulatory mechanisms remain poorly understood. In this study, we investigated the role of <i>PagARF3.1</i>, a homolog of <i>Arabidopsis</i> auxin response factor 3, in hybrid poplar (<i>Populus alba</i> × <i>Populus glandulosa</i> clone cv. '84K'), focusing on its involvement in adventitious root formation. GUS staining analysis revealed that <i>PagARF3.1</i> was expressed in adventitious root tips, pericycle cells, early root primordia, and outgrowing roots. Knockdown of <i>PagARF3.1</i> delayed adventitious root formation and reduced root biomass in transgenic plants, whereas overexpression of <i>PagARF3.1</i> promoted earlier rooting and increased the number of adventitious roots. Real-time quantitative polymerase chain reaction analysis indicated that the expression levels of <i>PagIPT5a</i> and <i>PagIPT5b</i> were significantly elevated in <i>PagARF3.1</i> RNAi lines and reduced in overexpression lines. Yeast one-hybrid assays and ChIP-PCR analysis demonstrated that PagARF3.1 directly binds to the promoter regions of <i>PagIPT5a</i> and <i>PagIPT5b</i>, thereby regulating their expression. These findings collectively demonstrate that PagARF3.1 acts as a positive regulator of adventitious root formation by repressing IPT-mediated cytokinin biosynthesis.