Functional characterization of NtHDZIPIV9 in regulating epidermal development in Nicotiana tabacum

The epidermis serves as the initial barrier between plants and their external environment, providing protection against factors such as water loss, temperature stress, and biological invasions. Despite its crucial role, the molecular mechanism of plant epidermal development is still not well understood. In this study, a novel member of the homeodomain leucine zipper Ⅳ family (NtHDZIPIV9) that plays a crucial role in the development of epidermal cuticles was identified in tobacco (Nicotiana tabacum L.). Subcellular localization analysis indicated the presence of NtHDZIPIV9 in the nucleus. Overexpression of NtHDZIPIV9 (NtHDZIPIV9-OE) resulted in increased cuticle thickness, pigment content, and leaf surface exudates. Conversely, these characteristics were significantly reduced in NtHDZIPIV9-knockout plants (NtHDZIPIV9-KO). RNA sequencing analysis of N. tabacum K326 (CK) and NtHDZIPIV9-KO plants revealed that NtHDZIPIV9 is involved in hormone regulation and defense responses. Further screening of differentially expressed transcription factors showed that NtHDZIPIV9 modulates the expression of nine genes (NtZFP8, NtATML1, NtARF3, NtMIXTA, NtTCP14, NtEXO1, NtSRA1, NtSHN1, and NtBDG) related to epidermal development. The yeast two-hybrid system was used to screen NtHDZIPIV9-interacting proteins and revealed potential interactions with twenty proteins, including six involved in epidermal development (NtGRP2, NtHDZIPIV12, NtHornerin-like, Ntpod, NtEXO1, and NtEXO2). Among these twenty candidate genes, the transcription levels of seven genes were significantly different in CK and NtHDZIPIV9-KO plants, including NtHDZIPIV12, Ntpod, NtEXO1, NtGRP2, NtCDR1, NtGLB1, and NtRBCS4. This study provides new insights into the possible mechanism underlying plant epidermal development, and lays a foundation for future studies in this area.