Two duplicated GhMML3 genes coordinately control development of lint and fuzz fibers in cotton

Cotton produces two types of fibers: fuzz and lint. Cotton yield is determined by the number of epidermal cells that develop into lint fibers. Despite numerous studies, the genetic and molecular mechanisms that control lint and fuzz fiber development remain unclear. Here, using the recessive naked-seed or fuzzless-linted mutant (n₂NSM) in combination with gene editing and complementation, we found that the recessive fuzzless gene n₂ encodes the MYBMIXTA-like (MML) transcription factor GhMML3_D12. Overexpression of GhMML3_D12 in n₂NSM restored fuzz fiber development, whereas CRISPR-Cas9 knockout of GhMML3_D12 in wild-type cotton (J668) resulted in a fuzzless-linted phenotype. Interestingly, simultaneous edits to GhMML3_D12 and its duplicate GhMML3_A12 resulted in plants with a fiberless (fuzzless-lintless) phenotype. Detailed investigation of the seed fiber phenotypes of segregating progeny derived from a cross between J668 and a fiberless gene-edited mutant of GhMML3 (#mml3s) not only identified progeny that mimicked natural fuzzless and fiberless mutants but also revealed that the duplicated GhMML3_A12 and GhMML3_D12 regulate the development of fuzz and lint fibers in a dose-dependent manner. Comparative transcriptome analysis and single-cell RNA sequencing identified GhMML3 as the central hub of the gene network that regulates fiber initiation and early-stage elongation. The gene regulatory network revealed potential candidate genes and key regulators that may contribute to fiber initiation and development, and a model for the control of lint and fuzz fiber development by GhMML3 was proposed. We also found that the GhMML3_D12 protein can bind directly to the promoters of GhHD-1 and GhMYB25, two key genes involved in fiber initiation, thereby activating their expression. This study provides new insights into the fundamental mechanisms that underlie cotton fiber development.