A HECT‐Type E3 Ubiquitin Ligase TaUPL21 Promotes Root Development in Caucasian Clover by Directly Activating Transcription of TaAGL29

Caucasian clover (Trifolium ambiguum M. Bieb.) is a valuable perennial legume with a robust root system. HECT-type E3 ubiquitin ligases (UPLs) are known regulators of plant growth, but their functions in root development, particularly in forage legumes, remain largely unexplored. Here, we identified 32 TaUPL genes in Caucasian clover and found that TaUPL21 is highly expressed in rhizome buds. Overexpression of TaUPL21 significantly enhanced root growth, including increases in root length, volume, and tip number. Strikingly, DNA affinity purification sequencing (DAP-Seq) revealed an unexpected DNA-binding capacity of TaUPL21. We further identified the MADS-box transcription factor TaAGL29 as a direct downstream target. Yeast one-hybrid (Y1H) and dual-luciferase (d-Luc) assays confirmed that TaUPL21 directly binds to the promoter of TaAGL29 and functions as a transcriptional activator. Our results unveil a novel, non-canonical function for a HECT E3 ubiquitin ligase, providing fresh insights into the functional versatility of E3 ubiquitin ligases and present a valuable genetic resource for improving root architecture in plants.