Glandular trichomes are universal epidermal structures that produce abundant specialized metabolites. However, knowledge of the initiation of glandular heads in glandular trichomes is limited. Herein, we found an intrinsic link of morphogenesis between glandular trichomes and non-glandular trichomes. Two novel homeodomain leucine zipper II members in tobacco (Nicotiana tabacum), NtHD9 and NtHD12, played important roles in long glandular trichome formation: NtHD9 was responsible for glandular head formation, while NtHD12 simultaneously controlled the formation of stalks and glandular heads. DAP-seq analysis suggested that NtHD9 can bind to the KKGCATTWAWTR motif of the cytochromes P450 94C1 (NtCYP94C1) promoter, which is involved in jasmonoyl-isoleucine oxidation. RNA-seq analysis of non-transformed tobacco and nthd9 plants revealed that NtHD9 modulates the expression of jasmonate (JA) signaling- and six trichome development-related genes. Notably, MeJA treatment restored the morphogenesis of long glandular trichomes in nthd9 and nthd12 plants, and the size of glandular heads increased with increasing MeJA concentration. However, the phenotype of long glandular trichome absence in double mutants of NtHD9 and NtHD12 could not be restored by MeJA. Our data demonstrate that NtHD9 and NtHD12 have distinct major functions yet overlapping roles in long glandular trichome formation via JA signaling.

Exogenous methyl jasmonate (MeJA) treatment induces glandular trichome development in Nicotiana benthamiana, but the function of JAZ proteins, acting as core repressors, and their downstream genes have not been clearly shown in plants. Here, a bioinformatics analysis of 71 JAZ genes from tobacco, Arabidopsis thaliana, and tomato was carried out and shown to share highly conserved domains. Then, the expression profile of 17 NbJAZs in different tissues was analyzed, and NbJAZ3 was highly expressed in trichome. Through transgenic technology, we demonstrated that the glandular trichome density of NbJAZ3‐overexpression lines significantly decreased with lower expression levels of NbWo, NbCycB2, and NbMIXTA. In contrast, the trichome density of NbJAZ3 RNAi lines slightly increased with higher expression level of NbWo. Given the negative protein feedback regulation relationship between NbCycB2 and NbWo, we verified that MeJA induced NbWo expression. NbWo was a direct target gene of NbJAZ3 and further demonstrated that NbJAZ3 inhibited the transcriptional activation of NbCycB2 by NbWo. Together, our findings outline a novel JA‐meditated glandular trichome development model consisting of the NbJAZ3‐NbWo‐NbCycB2 axis.