NtWUN Negatively Regulates Tobacco Trichome Development

doi:10.13560/j.cnki.biotech.bull.1985.2026-0339

Abstract

Abstract:

Objective This study aimed to elucidate the mode of action of the transcription factor NtWUN in mediating jasmonic acid (JA) signaling and to clarify the internal mechanism by which this transcription factor regulates trichome growth and development in tobacco. The findings would provide a theoretical and experimental basis for further dissecting the molecular mechanism underlying JA-mediated development of plant epidermal structures. Method In the common tobacco cultivar K326, multiple approaches including homologous cloning, bioinformatic analysis, subcellular localization assay, and quantitative real-time PCR (RT-qPCR) were adopted to characterize the sequence features and protein structure of the NtWUN gene, determine its subcellular localization properties, and clarify its tissue-specific expression patterns as well as transcriptional responses to methyl jasmonate (MeJA) treatments at varying concentrations. Furthermore, overexpression and knockout lines of NtWUN were generated to functionally validate the biological roles of this gene. Result NtWUN has two homologous sequences, NtWUN-1 and NtWUN-2. The coding region of NtWUN-1 is 630 bp in length, encoding 209 amino acids, while that of NtWUN-2 is 636 bp, encoding 211 amino acids. Phylogenetic analysis indicated that NtWUN-1 is highly homologous to Nicotiana sylvestris, and NtWUN-2 is highly homologous to N. tomentosiformis; both are closely related to homologous genes in Lycium barbarum. Expression pattern analysis showed that both NtWUN-1 and NtWUN-2 were most highly expressed in leaves, followed by trichomes, stems, and flowers, with the lowest expression in roots. Following treatment with different concentrations of MeJA, the relative expression levels of both genes initially increased and then decreased. Both genes were strongly induced by MeJA at 24 h, with expression levels upregulated by 10.2-fold and 11.8-fold, respectively. Subcellular localization results showed that NtWUN-1 and NtWUN-2 were both localized in the nucleus. Observation of trichome morphology and density revealed that the NtWUN overexpression line (OE8) showed 37.5% and 35.7% reductions in long-stalked and short-stalked glandular trichomes, respectively, while the knockout line (KO2) exhibited 50.0% and 57.1% increases in these two trichome types. The trichome types in the above transgenic lines were not affected. Conclusion The transcription factor NtWUN negatively regulates JA-mediated development of long-stalked and short-stalked glandular trichomes in tobacco.

Key words: tobacco, NtWUN, long-stalked trichome, short-stalked trichome, trichome density, gene editing, overexpression, jasmonic acid

LIU Na-wei, QIAN Meng-ying, ZHANG Hong-ying, WANG Zhao-jun, CUI Hong, YAN Xiao-xiao. NtWUN Negatively Regulates Tobacco Trichome Development[J]. Biotechnology Bulletin, 2026, 42(9): 128-136.

Figures/Tables 6

Fig. 1 Cloning and bioinformatics analysis of NtWUNA: PCR electrophoresis of NtWUN cloning. B: Phylogenetic tree of NtWUN and its homologous proteins. C: Multiple sequence alignment of NtWUN homologous proteins from closely related species. Nt: Nicotiana tabacum. Nsyl: N. sylvestris. Ntom: N. tomentosiformis. Na: N. attenuata. Lb: Lycium barbarum. Ca: Capsicum annuum. Sl: Solanum lycopersicum. St: Solanum tuberosum. Gh: Gossypium hirsutum. Ga: Gossypium arboreum

Fig. 2 Expression pattern of NtWUN in different tissues

Fig. 3 Expression analysis of NtWUN in response to treatments with different concentrations of MeJA

Fig. 4 Subcellular localization of the NtWUN protein

Fig. 5 Identification of NtWUN-overexpressing plants and homozygous knockout linesA: PCR identification of NtWUN overexpression positive lines (M: DNA marker 2 000; 1: ddH₂O; 2: NtWUN overexpression plasmid; 3-11: nine individual transgenic positive plants). B: Gene expression levels in NtWUN-2 overexpression positive plants (**P<0.01, with the expression level in K326 as reference 1). C: Sequencing analysis of the sgRNA region in homozygous knockout plants. D: Protein translation of NtWUN in homozygous mutant plants. Short horizontal lines within red boxes indicate premature translation termination

Fig. 6 Morphological observation (A) and density analysis (B) of trichomes on the leaf surfaceDifferent lowercase letters above the bars within the same group indicate significant differences at the 0.05 level

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