Transcriptional Activation of <i>NaNAC72</i> Suppresses Nicotine Biosynthesis in <scp>DNA</scp> Methyltransferase <i>NaDRM2‐like2</i> Mutants of <i>Nicotiana attenuata</i>

Nicotine, the most abundant alkaloid produced in Nicotiana species (Saitoh et al. 1985), plays a vital role in defence against insect herbivores (Steppuhn et al. 2004). Several transcription factors (TFs) have been identified as positive regulators, such as NtMYC2, NtERF189, NtERF199 and NtMYB305a (Shoji et al. 2010; Shoji and Hashimoto 2011; Sui et al. 2020; Qin et al. 2021; Bian et al. 2022). In contrast, very few negative regulators of nicotine biosynthesis have been reported. Hence, discovering new negative regulators is crucial for understanding how plants defend themselves against insect herbivores, and is also valuable for breeding tobacco varieties with increased nicotine content through mutation. DNA methylation is a conserved epigenetic modification involved in gene regulation. Interestingly, most root-specifically expressed nicotine-related genes share a DNA methylation valley as a common feature in wild tobacco Nicotiana attenuata (Tong et al. 2025). When DNA methyltransferase genes were silenced by virus-induced gene silencing (VIGS), the transcription levels of NaPMTs (the key enzyme genes for nicotine biosynthesis) and NaERF1-like (homologue of NtERF199) significantly decreased after silencing either NaCMT2 or NaDRM2-like2; however, increased after silencing NaCMT3-like1 or NaCMT3-like2 (Figure S1). These findings indicated that silencing DNA methyltransferases affects the expression of nicotine-related genes. Thus, investigating mutants of these genes may lead to the discovery of new negative regulators of nicotine. NaDRM2-like2 shows high expression in seeds but very low expression in leaves and roots (Figure S1). As the lowest expression level of NaPMTs, the key enzyme genes for nicotine biosynthesis, was detected in NaDRM2-like2-silenced plants (Figure S1), we therefore generated two NaDRM2-like2 mutants by CRISPR/Cas9 (Figure S2a). Both mutants had early termination of translation due to a frameshift mutation but grew as well as wild-type (WT) plants without any morphological abnormalities (Figure S2b). These mutants had lower transcription levels of nicotine-related genes, including NaPMTs, NaA622, and NaERF1-like, except NaBBLs (Figure 1a). Consequently, they produced less nicotine (Figure 1a) and were more vulnerable to the generalist insect herbivore Spodoptera littoralis (Figure S2c). To understand why less nicotine accumulated in drm2-like2 mutants, we conducted whole-genome bisulfite sequencing (WGBS) and RNA sequencing on WT and drm2-like2 mutant roots. WGBS results showed that the methylation level and pattern of nicotine-related genes, including NaPMT, NaBBLa, NaBBLb, and NaERF1-like, were similar in WT and mutants (Figure S3). Nevertheless, we successfully identified NaNAC72 as the only TF with both differentially methylated regions and differential expression in mutants compared to WT (Figure 1b). Bisulfite sequencing (BSP) confirmed the hypermethylation of the NaNAC72 promoter region in the drm2-like2 mutant (Figure 1c). Quantitative RT-PCR also confirmed that NaNAC72 expression was increased by more than 3-fold in drm2-like2 mutant roots (Figure 1d). Thus, our results indicated that an unusually high expression of NaNAC72 with hypermethylation of its promoter was detected in the drm2-like2 mutant. Furthermore, co-expression analysis revealed a strong negative correlation between the expression of NaNAC72 and root-specifically expressed nicotine genes (Figure S4a). The plant-specific NAC (NAM, ATAF1, 2 and CUC2) TF family is one of the largest TF families in plants. Currently, only one study has investigated the regulation of nicotine biosynthesis by NAC TFs, revealing that NtNAC-R1 positively regulates nicotine biosynthesis (Fu et al. 2013). No NAC TFs have yet been identified as negative regulators of nicotine biosynthesis. Two NaNAC72 knockout mutants were generated by the CRISPR/Cas9 system (Figure S4b). They had higher expression levels of nicotine-related genes, including NaPMTs, NaA622, NaBBLs and NaERF1-like. Importantly, nicotine content was increased by 35%–42% in both mutants (Figure 1e), without affecting plant growth and morphology (Figure 1f). The negative role of NaNAC72 was further supported by the NaNAC72-overexpression lines, which had a two- to three-fold increase in NaNAC72 expression in their roots (Figure S4c). Both overexpression lines decreased the expression of NaPMTs, NaA622, NaBBLs, NaERF1-like, NaMPO1 and NaQPT2, and reduced nicotine content (Figure S4c). Yet, the plant growth and morphology were similar to WT (Figure S4d). Electrophoretic mobility shift assays and chromatin immunoprecipitation coupled with quantitative PCR revealed the direct binding of NaNAC72 to the promoters of NaPMT1.1 and NaERF1-like (Figure S5 and Figure 1g), which are two key genes involved in the biosynthesis and regulation of nicotine. Further dual-luciferase assays indicated that the overexpression of NaNAC72 significantly decreased the promoter activity of NaPMT1.1 and NaERF1-like (Figure S5). In conclusion, our findings suggest that changes in DNA methylation affect the expression of nicotine-related genes, with distinct effects exerted by different DNA methyltransferases. Particularly, the NaDRM2-like2 mutation reduces nicotine production. Through DNA methylation and transcriptome profiling, we identified NaNAC72 as a new negative regulator of nicotine biosynthesis. It directly suppresses the transcriptional activity of nicotine-related genes, including NaPMT1.1 and NaERF1-like. Our results provide new insights into the regulation of defence metabolite by DNA methylation and establish NaNAC72 as a valuable breeding target for manipulation of nicotine production. J.W., L.W., and A.T. conceived the project, designed the experiments, and drafted the manuscript; A.T. performed most experiments and analyzed the data. R.L. generated the NaDRM2-like2 mutants by CRISPR/Cas9 technology. X.C. generated the NaNAC72 mutants and NaNAC72 overexpression plants. B.W. analyzed the DNA methylation results. WGBS and transcriptome data are available from Genome Sequence Archive under accession No. CRA08374. Other data are available within the article and its Supporting Information S1. Data S1: Supporting Information. Table S1: Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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