The R2R3-MYB transcription factor ScMYB20 negatively regulates drought and salt tolerance through a dual-repression of ScCHALCONE SYNTHASE-1 (ScCHS1)-mediated flavonoid biosynthesis in the desert moss Syntrichia caninervis.

The desert moss Syntrichia caninervis is one of the most desiccation-tolerant land plants known and provides a powerful system for dissecting the molecular foundations of extreme stress adaptation in early-diverging land lineages. The MYB transcription factor superfamily orchestrates secondary metabolism and stress signaling across plants, yet its lineage-specific evolution and mechanistic deployment in bryophytes remain poorly understood. Here, we identified 65 ScMYB genes in the S. caninervis genome and showed that the family expanded predominantly through dispersed duplication, with no detectable synteny to vascular-plant MYBs, indicating bryophyte-specific neo-functionalization. Integrating phylogenetic clustering, cis-element architecture and stress-responsive expression profiling, we pinpointed ScMYB20, a nuclear-localized, S13-subgroup R2R3-MYB that is rapidly and strongly induced by dehydration and salinity. Heterologous overexpression in Arabidopsis, together with overexpression and RNAi in S. caninervis, demonstrated that ScMYB20 negatively regulates drought and salt tolerance by suppressing antioxidant capacity, osmotic adjustment and photosynthetic performance, while concomitantly elevating ROS and MDA accumulation. Mechanistically, ScMYB20 directly binds a TAACCA motif in the ScCHS1 promoter to repress its transcription, and simultaneously sequesters the WD40 protein ScTTG1, a positive transcriptional activator of ScCHS1, thereby antagonising ScTTG1-mediated activation. Transient ScCHS1 overexpression restored flavonoid accumulation, antioxidant capacity and stress tolerance. Together, our findings define a dual-repression module (ScMYB20-ScTTG1-ScCHS1) that fine-tunes flavonoid flux under abiotic stress, and provide evolutionary and mechanistic insights into how R2R3-MYB repressors evolved to balance metabolic investment and stress survival in land plants.