Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome-wide association study (GWAS) on type-I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map-based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc-finger protein. Transgenic experiments showed that hair-absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type-I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein-protein interaction between them was further detected through pull-down and yeast two-hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.

SUMMARYCuticular waxes play a crucial role not only in plant defense against biotic and abiotic stresses, but also in the quality and storability of fruits, such as the tomato (Solanum lycopersicum). Although the biosynthetic pathways of waxes have been extensively characterized, the regulatory mechanisms underlying wax biosynthesis in tomato remain largely unclear. Here, we show that Woolly (Wo), a multicellular trichome regulator, is involved in modulating wax biosynthesis in tomato. Wo enhances the expression of the wax biosynthetic genes SlCER6, SlKCR1, and SlPAS2, and the wax transporter gene SlLTP, and thereby promotes wax accumulation. Furthermore, Wo directly binds to the L1‐box in the promoter of SlCER6, an essential element of the very‐long‐chain fatty acid elongase complex. Intriguingly, overexpression (OE) or knock‐down of SlMYB31, an MYB transcription factor that physically interacts with Wo in vivo and in vitro, produces marked changes in wax composition, and whereas Wo knock‐down inhibits wax accumulation in SlMYB31‐OE lines, SlMYB31 knock‐down inhibits wax accumulation in Wo‐OE lines, implying that these two genes function in the same pathway. Lastly, SlCER6 expression is induced by abscisic acid in a manner that is partially dependent on Wo. These results demonstrate that Wo and SlMYB31 cooperatively control tomato cuticular wax biosynthesis by regulating the expression of SlCER6.

Trichomes are specialized epidermal appendages that serve as an excellent model to study cell morphogenesis. Although the molecular mechanism underlying trichome morphogenesis in Arabidopsis has been well characterized, most of the regulators essential for multicellular trichome morphology remain unknown in tomato. In this study, we determined that the recessive hairless-2 (hl-2) mutation in tomato causes severe distortion of all trichome types along with increased stem fragility. Using map-based cloning, we found that the hl-2 phenotype was associated with a 100-bp insertion in the coding region of Nck-associated protein 1, a component of the SCAR/WAVE complex. Transgenic experiments confirmed that the hl-2 phenotype was caused by loss-of-function of this gene. Direct protein-protein interaction was detected between Hl-2 and Hl (SRA1) using yeast two-hybrid and co-immunoprecipitation assays, implying that these proteins may work together during trichome formation. In addition, knock-down of an HD-Zip IV transcription factor, HDZIPIV8, distorted trichomes similarly to the hl-2 mutant. HDZIPIV8 regulates the expression of Hl-2 by binding to the L1-box in the Hl-2 promoter region and is involved in organizing actin filaments. We also found that the brittleness of hl-2 stems resulted from decreased cellulose content. Taken together, these findings suggest that the Hl-2 gene plays an important role in controlling multicellular trichome morphogenesis and stem mechanical properties in tomato.

Using map-based cloning, we delimited the dialytic gene to an approximately 109-kb fragment, which controls multicellular trichome formation and stamen development in tomato. Trichomes exist in the epidermis of nearly all terrestrial plants, including unicellular and multicellular types. The molecular mechanism of unicellular trichomes in Arabidopsis is well characterized. However, knowledge about the regulatory pathway of multicellular trichomes in tomato (Solanum lycopersicum) is limited. Phenotypic analysis of the dialytic (dl) mutant LA3724 demonstrated that the trichomes are forked and the stamens are unclosed. To clone and characterize dl, we mapped this gene to an approximately 109-kb fragment using two F2 populations derived from the two crosses of dl mutant: LA3724 × IL8-1 and LA3724 × LA1589 (Solanum pimpinellifolium). Two types of molecular markers were utilized in this study, including cleaved amplified polymorphic sequences and insertion-deletion events. Sequence analysis predicted the presence of seven putative open reading frames, including two unknown proteins, two phospholipase Ds, glycosyl hydrolase family 5 protein/cellulose, choline/ethanolamine kinase, and aquaporin-like protein. The aquaporin-like protein gene was evidently upregulated in dl mutant. Thus, we inferred that this gene is a potential candidate for the phenotypes. The results provide a basis to elucidate the regulatory pathway responsible for trichome formation and stamen development in tomato.

Trichomes are specialized glandular or non-glandular structures that provide physical or chemical protection against insect and pathogens attack. Trichomes in Arabidopsis, as typical non-glandular structures, have been extensively studied. However, the molecular mechanism underlying glandular trichome formation and elongation still remains largely unknown. We previously demonstrated that Hair (H) is essential for the formation of type I and type VI trichomes. Here, we found that overexpression of H increased the density and length of tomato trichomes. We revealed that H physically interacts with its close homolog SlZFP8-like (SlZFP8L) and SlZFP8L also directly interacts with Woolly (Wo) by biochemical assays. SlZFP8L overexpression plants showed increased trichome density and length. We further found that the expression of SlZFP6, encoding a C2H2 zinc finger protein, is positively regulated by H. We identified that SlZFP6, is a direct target of H through ChIP-qPCR, Y1H, and LUC assays. Similar to H and SlZFP8L, the overexpression of SlZFP6 also increased the density and length of tomato trichomes. Taken together, our results suggest that H interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato.