Abstract: Drought severely constrains alfalfa (Medicago sativa L.) productivity, yet dissecting drought–response mechanisms in this important perennial forage crop remains challenging because of its highly heterozygous autotetraploid genome, the extended cycle required for stable transformation and regeneration, and the time-consuming evaluation of drought responses in mature plants. Here, we identified MsMYB71, a drought-responsive R2R3-MYB transcription factor, as a positive regulator of drought tolerance in alfalfa. Mechanistically, MsMYB71 directly bound to the promoter of MsPrxQ (peroxiredoxin Q), encoding a chloroplast-associated peroxiredoxin Q, and activated its expression, thereby promoting redox homeostasis under drought stress. In contrast, MsPRL1 (PLEIOTROPIC REGULATORY LOCUS 1, a WD40-repeat protein) and MsCUL4 (Cullin4, a Cullin-family scaffold protein) interacted with MsMYB71 and attenuated its protein accumulation and transcriptional output. To establish the physiological relevance of this regulatory module directly in alfalfa, we generated stable overexpression and RNA interference lines for all four genes. Genetic and physiological analyses showed that MsMYB71 enhanced drought tolerance and restricted excessive reactive oxygen species accumulation. Stable manipulation of MsPrxQ further established its positive role in drought tolerance and antioxidant defense. In contrast, MsPRL1 and MsCUL4 negatively regulated drought tolerance and physically interacted with MsMYB71. Their co-expression reduced MsMYB71 protein accumulation in a proteasome-sensitive manner and attenuated MsMYB71-dependent activation of MsPrxQ, consistent with an MsPRL1/MsCUL4-associated attenuation mechanism. Together, our findings define an MsPRL1/MsCUL4–MsMYB71–MsPrxQ module that couples transcriptional activation with protein-level attenuation to fine-tune redox homeostasis and drought adaptation in alfalfa.

Key words: drought tolerance, Medicago sativa, MYB transcription factor, redox homeostasis