The R2R3-MYB transcription factor GhMYB35 governs anther development and pollen viability in upland cotton

Male reproductive development is fundamental to the life cycle of flowering plants, culminating in seed production. Aberrations in anther development frequently lead to male sterility, yet the underlying molecular mechanisms in upland cotton (Gossypium hirsutum) remain largely uncharacterized. The R2R3-MYB family of transcription factors are known key regulators of diverse developmental processes, including male fertility in several model species. Here, we identify and functionally characterize GhMYB35, an R2R3-MYB transcription factor that plays an essential role in cotton anther development. CRISPR/Cas9-mediated knockout of GhMYB35 resulted in complete male sterility, with mutants (ghmyb35) exhibiting indehiscent anthers, shorter filaments, and a total absence of viable pollen. Expression analyses reveal that GhMYB35 is predominantly expressed in anthers, with peak expression of its A- and D-subgenome homoeologs occurring at developmental stage 7. Subcellular localization results show that bothGhMYB35_AandGhMYB35_Dare nuclear-localized transcription factors. Furthermore, the total absence of GhMYB35 leads to pollen abortion and subsequent anther collapse without dehiscence. Collectively, our findings establish GhMYB35 as a critical regulator of anther maturation, thereby elucidating a key component of the molecular network governing male fertility in cotton.