taxonID	type	description	language	source
03F987D2FFF8FFF3AE7BFE06FF01FE27.taxon	description	In this study, BLASTP and HMM searches using the NAC protein sequences of Arabidopsis as query were performed to broadly identify S. miltiorrhiza NAC family members. A total of 84 NAC proteins were identified in the S. miltiorrhiza genome. They were named Sm- NAC 4 - Sm- NAC 84 is based in accordance with gene IDs in the newly sequenced genome. Sm- NAC 1 - Sm- NAC 3 had been identified previously (Zhang and Liang, 2019; Yin et al., 2020; Zhu et al., 2019 b); therefore, their names were maintained (Supplementary Table S 1). The coding DNA and protein sequences of Sm-NAC gene family members are given in Supplementary Files S 1 – S 2. The protein sequence lengths of the 84 Sm-NACs are quite different (Supplementary Table S 1). The longest is 794 bp (Sm-NAC 79), and the shortest is only 120 bp (Sm-NAC 27). The molecular weights range from 14.01 kDa (Sm-NAC 27) to 89.74 kDa (Sm-NAC 79), and the isoelectric points (pI) range from 4.53 (Sm-NAC 83) to 9.73 (Sm-NAC 4). The locations of the 84 Sm - NAC genes on the chromosomes of S. miltiorrhiza are shown in Supplementary Figure S 1, and all 84 Sm - NAC genes, except for Sm - NAC 4 that is on the scaffold, are distributed among the eight chromosomes. There was no significant correlation between the number of Sm - NAC genes present per chromosome and the chromosomal length. To investigate the phylogenetic relationships among the 84 Sm-NACs, a phylogenetic tree was constructed by combining Sm-NACs with Arabidopsis NAC proteins (AtNACs). The 84 Sm-NACs were divided into nine families (Groups 1 – 9) (Fig. 1). There was an unequal distribution of Sm-NACs among the groups, with the Group 9 subfamily, containing 22 genes, having the most members, followed by Groups 7 and 6, each containing 15 proteins. The Group 3 subfamily had the least members, containing only two genes.	en	Zhang, Haihua, Xu, Jinfeng, Chen, Haimin, Jin, Weibo, Liang, Zongsuo (2021): Characterization of NAC family genes in Salvia miltiorrhiza and NAC 2 potentially involved in the biosynthesis of tanshinones. Phytochemistry (112932) 191: 1-8, DOI: 10.1016/j.phytochem.2021.112932, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112932
03F987D2FFFDFFF5AD2DFC0FFAADFA32.taxon	description	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, NAC 042 (JUB 1) directly represses the hormone biosynthetic genes GA 3 ox 1 and DWARF 4 (DWF 4), leading to typical GA / BR deficiency phenotypes in A. thaliana (Shahnejat-Bushehri et al., 2016). In Foxtail millet (Setaria italica L.), SiNAC 1 positively regulates leaf senescence and is involved in a positive feedback loop via ABA biosynthesis (Ren et al., 2018); In Oryza sativa, OsNAC 2 affects the expressions levels of auxin- and cytokinin-responsive genes to regulate root development (Mao et al., 2020). The A. thaliana NAC family proteins ANAC 019 and ANAC 055, 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 - NAC 2, was selected for further studies on gene function. Sm - NAC 2 - 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, MdNAC 52 regulates anthocyanin and proanthocyanidin biosynthesis (Sun et al., 2019 b). The OsSWNs and ZmSWNs NACs, regulate the ectopic depositions of cellulose, xylan, and lignin (Zhong et al., 2011). The ANAC 078 protein is involved in flavonoid biosynthesis, and its expression leads to anthocyanin accumulation (Morishita et al., 2009). BoNAC 019 negatively regulates anthocyanin biosynthesis in Arabidopsis (Wang et al., 2018 a). Thus, NAC genes play very important regulatory roles in plant secondary metabolic biosynthesis. 3.2. Possible Sm-NAC 2 - 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- NAC 1 plays a crucial role in UV-B irradiation-induced SalA biosynthesis (Zhu et al., 2019 a). 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 - NAC 2 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, MdNAC 52 binds to the MdMYB 9 and MdMYB 11 promoters increase anthocyanin and proanthocyanidin biosynthesis (Sun et al., 2019 b). The OsSWN and ZmSWN NACs in rice and maize, respectively, bind the OsMYB 46 and ZmMYB 46 promoters, respectively, and activate target genes to regulate the ectopic deposition of cellulose, xylan, and lignin (Zhong et al., 2011). NAC 2 binds to the CATGTG and CATGTC motifs present in the promoters of theMYB 2, Sm-MYB 98, MYB 11, MYB 9, and MYB 9 b transcription factors, which participate in tanshinone biosynthesis. In this study, we functionally determined that NAC 2 was a negative regulatory transcription factor of tanshinones. Whether Sm-NAC 2 - binding sites exist on Sm-MYB promoters require further experimental investigation.	en	Zhang, Haihua, Xu, Jinfeng, Chen, Haimin, Jin, Weibo, Liang, Zongsuo (2021): Characterization of NAC family genes in Salvia miltiorrhiza and NAC 2 potentially involved in the biosynthesis of tanshinones. Phytochemistry (112932) 191: 1-8, DOI: 10.1016/j.phytochem.2021.112932, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112932
03F987D2FFFCFFF7AD2DFF66FF68FD12.taxon	description	The assembly and annotation data of Salvia miltiorrhiza Bunge (Lamiaceae) in the Genome Warehouse in BIG Data Center under Project numbers PRJCA 003150, which are accessible at https: // bigd. big. ac. cn / gwh (Song et al., 2020). The Arabidopsis NAC amino acid sequences were obtained from TAIR (http: // www. arabidopsis. org) and were used as query in searches against the S. miltiorrhiza genome database using the BLASTP program to obtain homologous sequences (Jin et al., 2020). The Hidden Markov Model (HMM) corresponding to the NAC domain (PF 02365) was downloaded from the pfam protein database, and HMMER 3.2 was used to examine the NAC genes from the BLASTP aligned sequences. Default parameters were employed, and the cutoff value was set to 0.01 (Letunic and Bork 2018). Genes encoding proteins containing NAC domains were identified as NAC genes. All the Sm - NACs were mapped to the eight chromosomes and one scaffold of S. miltiorrhiza using the TBtools program and the physical locational information from the S. miltiorrhiza genome (Li et al., 2020 a). A multi-sequence alignment of NAC proteins from Arabidopsis and S. miltiorrhiza was performed using ClustalW in MEGA 7.0 with default parameters (https: // www. megasoftware. net /). Because the Sm-NAC family sequence lengths varied greatly, the alignment results were used to construct a phylogenetic tree using the Maximum Likelihood method with 1000 bootstrap replicates (Felsenstein 1985; Jones et al., 1992). Additionally, Evolview (http: // www. evolgenius. info /) was used to beautify the evolutionary tree (Subramanian et al., 2019). 5.2. Gene structure and protein motif analyses of Sm-NAC genes An online program of the gene structure display server (GSDS 2.0) (http: // gsds. cbi. pku. edu. cn / index. php) was used to draw the exonintron distribution of each Sm - NAC gene by comparing predicted coding sequences (Hu et al., 2015). Conserved motifs of Sm-NAC protein sequences were investigated using the online software MEME 5.0.4 (htt p: // meme-suite. org / tools / meme) with default values for the motif parameters and the number of motifs searched set as 20 (Bailey et al., 2009; Munir et al., 2020). TBtools was used to visualize the results (Chen et al., 2020).	en	Zhang, Haihua, Xu, Jinfeng, Chen, Haimin, Jin, Weibo, Liang, Zongsuo (2021): Characterization of NAC family genes in Salvia miltiorrhiza and NAC 2 potentially involved in the biosynthesis of tanshinones. Phytochemistry (112932) 191: 1-8, DOI: 10.1016/j.phytochem.2021.112932, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112932
03F987D2FFFCFFF7AD2DFF66FF68FD12.taxon	materials_examined	5.3. Expression profile analysis using transcriptome data To understand NAC gene expression changes after MeJA exposure in S. miltiorrhiza and the expression levels in different tissue, the transcriptome data was retrieved from the NCBI Sequence Read Archive. For the different tissues, we selected three tissues in the same batch of Sequence Read Archive (SRA) data: flower, leaf, and root (accession numbers: SRR 1020591, SRR 1043998, and SRR 1045051) (Chen et al., 2014). The transcriptome data after the MeJA treatment was selected from two-month-old sterile seedlings grown on 0.5 × MS medium containing 100 μM MeJA or the simulated solution (ethanol). The roots of the treated seedlings were collected from three biological replicates (accession numbers: SRR 11484256 - SRR 11484259, SRR 11484266, and SRR 11484271) (Zhou et al., 2020). In accordance with the TopHat BAM files and the reference GTF file, cuffdiff was used to calculate fragments per kb per million reads values (FPKM) of different tissue samples and MeJA-treated samples, and the expression differences between different samples were determined at the same time (Sulayman et al., 2019).	en	Zhang, Haihua, Xu, Jinfeng, Chen, Haimin, Jin, Weibo, Liang, Zongsuo (2021): Characterization of NAC family genes in Salvia miltiorrhiza and NAC 2 potentially involved in the biosynthesis of tanshinones. Phytochemistry (112932) 191: 1-8, DOI: 10.1016/j.phytochem.2021.112932, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112932
03F987D2FFFCFFF7AD2DFF66FF68FD12.taxon	description	5.4. RNA isolation and qRT-PCR Total RNA was extracted from the rhizome, leaves, and transgenic roots of S. miltiorrhiza in accordance with the instructions of the polysaccharide and polyphenol plant RNAprep Pure Plant Kit (TIANGEN, China). The RNA from the rhizome, leaves, and transgenic roots of S. miltiorrhiza were mixed and reverse transcribed into cDNA using a PrimeScript ™ II 1 st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). The cDNA was used as the template to amplify the NAC 2 gene for cloning. The cDNA for qRT-PCR was synthesized using the PrimeScript ™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Tokyo, Japan) with oligo dT. The qRT-PCR was performed in accordance with the instructions of the PrimeScript ™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa on a QuantStudio ™ 6 Flex (Life Technologies, Carlsbad, CA, USA )). The procedure was as follows: 95 ◦ C for 30 s, then 40 cycles of 95 ◦ C for 5 s and 59 ◦ C for 30 s. Each reaction was repeated three times. The reference gene was the actin (Yang et al., 2010). The primers used are list in Supplementary Table S 2. The 2 ΔΔ Ct method was used to analyze the qRT-PCR data and calculate the relative expression levels (Livak and Schmittgen, 2001). 5.5. Plant expression vector construction To construct the Sm - NAC 2 - overexpression vector, the gene-specific primers Sm-NAC 2 - OE-F and Sm-NAC 2 - OE-R were used to amplify the complete ORF of Sm - NAC 2. Gateway technology was used to construct the expression vector. First, the ORF of Sm - NAC 2 was cloned into the pDONR 207 entry vector using the BP Clonase Enzyme Kit, and then, it was cloned into the pK 7 WG 2 R destination vector using an LR Clonase Enzyme Kit (Invitrogen, MA, USA) (Ding et al., 2017). A 116 - bp sequence was amplified using the primers Sm-NAC 2 - RNAi- F and Sm-NAC 2 - RNAi-R to construct the plant RNAi vector. The amplified fragment was cloned into the pDONR 207 entry vector, and then cloned into the pK 7 GWIWG 2 R binary vector, as described by (Ding et al., 2017). The recombinant vector was confirmed by sequencing. The primers used in this experiment are provided in Supplementary Table S 2. 5.6. Acquisition of S. miltiorrhiza transgenic transgenic roots The leaves of the sterile seedlings of S. miltiorrhiza were cut into small pieces of 1 × 1 cm and placed them on a 1 / 2 MS solid medium for cultivating in the dark for 2 – 3 days at 25 ◦ C. Single colonies of the A. rhizogenes cells harboring the recombinant plasmid were inoculated into 50 ml of liquid YEB medium with 50 mg l 1 of spectinomycin, and grown on a shaker at 28 ◦ C for 16 – 18 h until the OD 600 nm reached 0.6. Cells were collected by centrifugation, and re-suspended in 50 ml of liquid 1 / 2 MS medium. Next, the leaf discs were submerged and shaken in the suspension for 30 min with 100 rpm at 25 ◦ C. Then, the leaf discs were taken out and cultured on 1 / 2 MS solid medium for 3 d in the dark. The leaf discs were moved onto 1 / 2 MS selection solid medium with 50 mg l 1 kanamycin and reduced cefotaxime. The sterilization medium was changed once in 10 – 15 d, and the concentration of cefotaxime in the medium was gradually reduced: from 500 mg l 1 to zero. When the transgenic roots grew to 4 – 5 cm, and a single root was cut from the leaf and placed on a sterile medium for individual culture. The rapidly growing kanamycin-resistant and agrobacterium-free transgenic roots were transferred to 50 ml of liquid 1 / 2 MS medium and maintained by transferring 0.3 g of root material into fresh 1 / 2 MS medium every 30 d (Ru et al., 2016). The WT control was transgenic roots developed using A. rhizogenes ATCC 15834 not harboring the plasmid. The genomic DNA from fresh transgenic roots was isolated use the cetyltrimethylammonium bromide (CTAB) method (Sambrock and Russel 2001). Four pairs of specific primers were used to identify positive transgenic strains (Supplementary Table S 2). The identified transgenic transgenic roots were cultured as described previously to further study Sm - NAC 2 functions (Zhang et al., 2020). 5.7. Extraction and determination of tanshinones The sampled transgenic roots were placed in an oven at 45 ◦ C until they were completely dehydrated, and then, the dried S. miltiorrhiza samples were crushed into a powder using a grinder. In total, 0.02 g of sample powder was placed into 2 mL of 70 % methanol. After soaking overnight in the dark, the sample was subjected to ultrasound for 45 min and then centrifuged at 8000 g for 10 min. The supernatant was removed and filtered through a 0.45 μm membrane. Afterward, 10 μL of the sample was used for HPLC detection on a Waters HPLC e 2695 system (Waters, Milford, MA, USA). The HPLC conditions were those established previously in our laboratory (Zhang et al., 2020). 5.8. Data statistics and analysis All the experiments were performed three times, and summary statistics are presented as means ± standard deviations (SDs). One-way ANOVAs (followed by a Tukey’ s comparisons) were used to test for significant differences among the means (indicated by different letters at P <0.05).	en	Zhang, Haihua, Xu, Jinfeng, Chen, Haimin, Jin, Weibo, Liang, Zongsuo (2021): Characterization of NAC family genes in Salvia miltiorrhiza and NAC 2 potentially involved in the biosynthesis of tanshinones. Phytochemistry (112932) 191: 1-8, DOI: 10.1016/j.phytochem.2021.112932, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112932
