Two duplicated GhMML3 genes coordinately control cotton lint and fuzz fiber development

Cotton produces fuzz and lint two types of fibers. Cotton yield is determined by the number of epidermal cells that develop into lint fibers. Despite numerous studies, the genetic and molecular mechanisms controlling lint and fuzz fiber development remain elusive. Here, using the recessive naked seed or fuzzless-linted mutant (n2NSM) in combination with gene editing and complement, we identified the recessive fuzzless gene n2 encodes an MML transcription factor, GhMML3_D12. Over-expressing GhMML3_D12 in n2NSM restored fuzz fiber development. Conversely, knocking out GhMML3_D12 in wild-type cotton (J668) by CRISPR/Cas9 led to a fuzzless-linted phenotype. Interestingly, simultaneous editing of duplicated GhMML3s (GhMML3_A12 and GhMML3_D12) resulted in plants with the fiberless (fuzzless-lintless) phenotype. Detailed investigation of the seed fiber phenotype of the segregating progeny derived from a cross between a fibreless mutant of GhMML3 (#mml3s) created by gene editing and J668 not only identified progeny mimicking the 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 dosage-dependent manner. Comparative transcriptome and single cell RNA-seq illuminated GhMML3 being the hub gene of the gene network regulating fiber initiation and early-stage elongation. The gene regulatory network revealed potential candidate genes and key regulators that may contribute to fiber initiation development. A model for lint and fuzz fiber development controlled by GhMML3 is proposed. We also revealed that GhMML3_D12 protein can directly bind to the promoters of GhHD-1 and GhMYB25, two key genes involved in fiber initiation, thereby activating their expression. The findings of this study provide fresh insights on the fundaments responsible for cotton fiber development.