Identification of a cis-element for long glandular trichome-specific gene expression, which is targeted by a HD-ZIP IV protein

Glandular trichomes are epidermal outgrowths that secret a variety of secondary metabolites, which not only help plants adapt to environmental stresses but also have important commercial value in fragrances, pharmaceuticals, and pesticides. In Nicotiana tabacum , it has been confirmed that a B-type cyclin, CycB2, negatively regulates the formation of long glandular trichomes (LGTs). This study aimed to identify the upstream regulatory gene involved in LGT formation by screening LGT-specific cis -elements within the NtCycB2 promoter. Using GUS as a reporter gene, the tissue-driven ability of NtCycB2 promoter showed that NtCycB2 promoter could drive GUS expression specifically in LGTs. Function analysis of a series of successive 5 ' truncations and synthetic segments of the NtCycB2 promoter indicated that the 87-bp region from -1221 to -1134 of the NtCycB2 promoter was required for gene expression in LGTs, and the L1-element (5 ' -AAAATTAATAAGAG-3 ' ) located in the 87-bp region contributed to the gene expression in the stalk of LGTs. Further Y1H and LUC assays confirmed that this L1element exclusively binds to a HD-Zip IV protein, NtHD13. Gene function analysis revealed that NtHD13 positively controlled LGT formation, as overexpression of NtHD13 resulted in a high number of LGTs, whereas knockout of NtHD13 led to a decrease in LGTs. These findings demonstrate that NtHD13 can bind to an L1element within the NtCycB2 promoter to regulate LGT formation.