Flavonoid metabolism enzymes GhCHS and GhDFR influence fiber elongation and secondary cell wall synthesis in cotton

The flavonoid metabolic pathway plays a pivotal role in plant growth, development, and environmental adaptation, and has undergone significant selection during cotton (Gossypium hirsutum) domestication. However, the functional role of flavonoid metabolism in fiber development remains poorly understood. In this study, we explore its functional significance in cotton fiber development by identifying expression quantitative trait loci (eQTLs) that regulate flavonoid biosynthesis and fiber-related traits. Through integrative eQTL mapping and transcriptomic analyses, we identified regulatory variants associated with key transcription factor and biosynthesis genes, including GhMYB46, GhMYB111, chalcone synthase (GhCHS) and dihydroflavonol 4-reductase (GhDFR). Functional characterization revealed that GhCHS and GhDFR play distinct roles in fiber development: in GhCHS-RNAi lines, excessive reactive oxygen species (ROS) accumulation during fiber elongation impaired cell expansion, resulting in significantly shorter fibers; in contrast, GhDFR suppression disrupted cellulose biosynthesis and secondary cell wall thickening, leading to fibers with increased micronaire values. Together, these findings indicate that eQTL-mediated regulatory variation reshapes the expression of flavonoid pathway genes, thereby influencing both fiber elongation and secondary cell wall formation. Such regulatory remodeling underscores the functional integration of flavonoid metabolism into fiber development and highlights its potential as a valuable genetic resource for future fiber improvement.