SlH3 and SlH4 promote multicellular trichome formation and elongation by upregulating woolly in tomato

Trichomes are tiny outgrowths on the plant epidermis that serve defensive purposes against various stresses. While the regulatory mechanisms underlying unicellular trichome development are well understood, those governing multicellular trichome formation remain largely unexplored. In this study, we reveal a new regulatory pathway involving the Hair3 (H3) and H4 genes, which encode C2H2 zinc finger proteins that participate in multicellular trichome development in tomato (Solanum lycopersicum). Using CRISPR-Cas9 to generate single- and double-knockout lines, we found that h3 and h4 single-mutant plants did not show altered trichome characteristics compared to wild-type plants. However, h3/h4 double-knockout plants displayed decreased densities of Types I, VI, and VII trichomes, increased densities of Types III and V trichomes, and reduced leaf and stem lengths of Type I trichomes, revealing that H3 and H4 redundantly regulate trichome development. Notably, protein interaction assays demonstrated that H3 and H4 formed both homo- and heterodimers, supporting their cooperative role. Transcriptome and gene expression analyses identified H3 and H4 as key regulators of several genes involved in trichome development, including Woolly (Wo) and its downstream targets, such as Wox3b, MX1, H, and HD8. Protein-promoter assays showed that H3 and H4 did not directly bind to the Wo promoter but rather interacted with Wo, thereby enhancing the expression of Wo and Wox3b. These findings establish H3 and H4 as key regulators of trichome development and provide novel insights into the mechanisms controlling multicellular trichome development in tomato plants.