3.1. Sm-NAC responds to jasmonic acid signals and participates in regulating the secondary metabolism of S. miltiorrhiza

The NAC gene family regulates multiple aspects of plant growth, development, plant hormone signaling, and secondary metabolism. NACs respond to hormone signals and regulate their biosynthesis. For example, NAC042 (JUB1) directly represses the hormone biosynthetic genes GA3ox1 and DWARF4 (DWF4), leading to typical GA/BR deficiency phenotypes in A. thaliana (Shahnejat-Bushehri et al., 2016). In Foxtail millet (Setaria italica L.), SiNAC1 positively regulates leaf senescence and is involved in a positive feedback loop via ABA biosynthesis (Ren et al., 2018); In Oryza sativa, OsNAC2 affects the expressions levels of auxin- and cytokinin-responsive genes to regulate root development (Mao et al., 2020). The A. thaliana NAC family proteins ANAC019 and ANAC055, as the transcription activators, regulate JA-induced expression of defense genes (Bu et al., 2008). In this study, a transcriptome data analysis revealed that 8 Sm -NAC s were significantly upregulated in response to MeJA signal. One of them, Sm -NAC2, was selected for further studies on gene function. Sm -NAC2 -overexpression lines inhibited tanshinone biosynthesis, whereas RNAi transgenic hairy-root lines promoted significantly tanshinone biosynthesis. NACs regulate the secondary metabolism of other plants. For example, MdNAC52 regulates anthocyanin and proanthocyanidin biosynthesis (Sun et al., 2019b). The OsSWNs and ZmSWNs NACs, regulate the ectopic depositions of cellulose, xylan, and lignin (Zhong et al., 2011). The ANAC078 protein is involved in flavonoid biosynthesis, and its expression leads to anthocyanin accumulation (Morishita et al., 2009). BoNAC019 negatively regulates anthocyanin biosynthesis in Arabidopsis (Wang et al., 2018a). Thus, NAC genes play very important regulatory roles in plant secondary metabolic biosynthesis.

3.2. Possible Sm-NAC2-associated regulatory mechanism of tanshinone biosynthesis

S. miltiorrhiza is an important bulk medicinal material. Tanshinones and salvianolic acids, as the main secondary metabolites, are the main active ingredients in S. miltiorrhiza and play important roles in the treatment of cardiovascular and cerebrovascular diseases. Sm- NAC1 plays a crucial role in UV-B irradiation-induced SalA biosynthesis (Zhu et al., 2019a). A total of 84 NAC transcription factors were identified in the S. miltiorrhiza genome, but the functions of these NACs in the regulation of secondary metabolites has not been widely studied in S. miltiorrhiza. Here, we found that Sm -NAC2 is a novel negative regulator of tanshinone biosynthesis in S. miltiorrhiza. As transcription factors, NACs regulate tanshinone biosynthesis either by regulating other transcription factors or by regulating structural genes. NACs act upstream of MYB. In apple, MdNAC52 binds to the MdMYB9 and MdMYB11 promoters increase anthocyanin and proanthocyanidin biosynthesis (Sun et al., 2019b). The OsSWN and ZmSWN NACs in rice and maize, respectively, bind the OsMYB46 and ZmMYB46 promoters, respectively, and activate target genes to regulate the ectopic deposition of cellulose, xylan, and lignin (Zhong et al., 2011). NAC2 binds to the CATGTG and CATGTC motifs present in the promoters of theMYB2, Sm-MYB98, MYB11, MYB9, and MYB9b transcription factors, which participate in tanshinone biosynthesis. In this study, we functionally determined that NAC2 was a negative regulatory transcription factor of tanshinones. Whether Sm-NAC2-binding sites exist on Sm-MYB promoters require further experimental investigation.