GhHDZ76, a cotton HD-Zip transcription factor, involved in regulating the initiation and early elongation of cotton fiber development in G. hirsutum

In this study, the whole HD-Zip family members of G. hirsutum were identified, and GhHDZ76 was classified into the HD-Zip IV subgroup. GhHDZ76 was predominantly expressed in the 0-5 DPA of fiber development stage and localized in the nucleus. Overexpression of GhHDZ76 significantly increased the length and density of trichomes in Arabidopsis thaliana. The fiber length of GhHDZ76 knockout lines by CRISPR/Cas9 was significantly shorter than WT at the early elongation and mature stage, indicating that GhHDZ76 positively regulate the fiber elongation. Scanning electron microscopy showed that the number of ovule surface protrusion of 0 DPA of GhHDZ76 knockout lines was significantly lower than WT, suggesting that GhHDZ76 can also promote the initiation of fiber development. The transcript level of GhWRKY16, GhRDL1, GhEXPA1 and GhMYB25 genes related to fiber initiation and elongation in GhHDZ76 knockout lines were significantly decreased. Yeast two-hybrid and Luciferase complementation imaging (LCI) assays showed that GhHDZ76 can interact with GhWRKY16 directly. As a transcription factor, GhHDZ76 has transcriptional activation activity, which could bind to L1-box elements of the promoters of GhRDL1 and GhEXPA1. Double luciferase reporter assay showed that the GhWRKY16 could enhance the transcriptional activity of GhHDZ76 to pGhRDL1, but it did not promote the transcriptional activity of GhHDZ76 to pGhEXPA1. GhHDZ76 protein may also promote the transcriptional activity of GhWRKY16 to the downstream target gene GhMYB25. Our results provided a new gene resource for fiber development and a theoretical basis for the genetic improvement of cotton fiber quality.