Salvia miltiorrhiza Bunge

Field: Agricultural and Biological Sciences (Life Sciences)

2.4. SmMAPK3 regulates the production of phenolic acids in S. miltiorrhiza To determine whether SmMAPK3 facilitates the biosynthesis of phenolic acids in S. miltiorrhiza, plasmids that contain the SmMAPK3 full-length ORF or empty vector (under the control of the CaMV35S promoter) were used to generate the respective transgenic plantlet lines. Positive transgenic plantlets were identified by PCR using gene-specific primers (Supplementary Table S2) to amplify the gene sequences containing the partial CaMV35S promoter and SmMAPK3 gene. In total, 28 SmMAPK3 -overexpressing (OM) and 16 empty vector control (EV) plantlet lines were generated. qRT–PCR analysis revealed that SmMAPK3 transcripts in the OM lines displayed an accumulation> 15- 25-fold greater than the vector control (Fig. 4). Three efficiently overexpressed genes were selected for follow-up functional analysis (named OM11, OM52, and OM87). HPLC analysis clearly indicated that the contents of RA and Sal B markedly increased in the OM lines (Fig. 4). In summary, these results indicated that transgenic plantlets overexpressing SmMAPK3 exhibit a promoting phenotype in terms of their biosynthesis of phenolic acids, indicating that SmMAPK3 operates as a positive regulator of phenolic acids in S. miltiorrhiza biosynthesis. We used qRT–PCR to determine the transcription levels of genes (TAT1, HPPR1, PAL1, C4H1, 4CL1, RAS 1, RAS 6 and CYP98A14) involved in phenolic acid biosynthesis in the OM and EV plantlets. Expression levels of the enzymes that we detected were significantly increased in the OM lines compared to the EV lines (Fig. 4), consistent with the increased accumulation pattern of phenolic acids (Fig. 4). These results suggest that SmMAPK3 positively controls phenolic acid biosynthesis in S. miltiorrhiza. 2.5. Potential upstream kinases of SmMAPK3 To clarify the upstream kinase of SmMAPK3, a Y2H assay was performed to identify upstream kinase candidates involved in the biosynthesis of phenolic acids. The Y2HGold strain yeast cells transformed with pGADT7- SmMAPKK7 and pGBKT7- SmMAPK3, pGADT7- SmMAPKK5 and pGBKT7- SmMAPK3, pGADT7- SmMAPKK4 and pGBKT7- SmMAPK3 as well as pGADT7- SmMAPKK2 and pGBKT7- SmMAPK3 grew on SD-LWHA with AbA and turned blue in the presence of the X-α- gal substrate (Fig. 5A). Then, we further verified the interaction between SmMAPK3 and protein members (SmMAPKK7, SmMAPKK5, SmMAPKK4 and SmMAPKK2) by performing an LCI assay. We used the empty vector with N-terminal domains and another empty vector with C-terminal domains together with every cLUC-fusion protein and nLUC-fusion protein construct as negative controls (Fig. 5B–C). As shown in Fig. 5D, strong fluorescent signals were detected when nLUC-SmMAPK3 was coexpressed with cLUC-SmMAPKK2, cLUC-SmMAPKK4, cLUC-SmMAPKK5, and cLUC-SmMAPKK 7 in tobacco (N. benthamiana) leaves. These findings demonstrate that SmMAPKK2/4/5/7-SmMAPK3 forms a cascade. 2.6. Protein–protein interaction involving SmMAPK3 To clarify the potential operating system controlled by SmMAPK3, a Y2H assay was performed to discover interacting protein candidates involved in the biosynthesis of phenolic acids. Using full-length SmMAPK3 as bait, the yeast prey cDNA library (samples containing several treatments (SA, MeJA and YE) and organs (roots, stem and leaves)) was screened. Ultimately, tens of candidate proteins, SmWRKYs, SmPRs, SmSTH, SmWHY, SmERFs, SmARF, SmIAAs, SmABP, SmGH3, SmABCG, SmPP2C, SmPYL, SmCOP, SmJAZ, SmTCP, SmbZIP, SmbHLHs, SmMYBs, SmNAC, SmTTG and SmWD40, were screened from the cDNA library (detailed information is listed in Supplementary Table S1, and sequences are listed in the Supplementary File). We divided the candidate proteins into nine groups: SA-related, ETH-related, auxin-related, CK-related, ABA-related, GA-related, JArelated, SL-related and other TFs. To further investigate the interactions between SmMAPK3 and the candidate proteins, we cloned the complete ORF sequences of genes of interest and re-examined them using Y2H. For other TFs, ETH-related, GA-related, ABA-related and auxin-related, we picked SmbZIP16, SmERF9, SmDELLA, SmPP2C14, SmARF7 and IAA1/9/14 separately (Supplementary Fig. S1). For SArelated and JA-related genes, we verified the other members of the signalling pathway, such as SmNPRs, SmTGAs, SmWRKYs, SmMYC2 and SmJAZs, in addition to the genes we screened (Supplementary Fig. S2 and Fig. 6A). Finally, the results in Supplementary Fig. S1 and Fig. 6A show that SmPP2C14, SmIAA9/14 and SmJAZ1-10 interact with SmMAPK3, and these proteins were taken as candidate partners of SmMAPK3. Surprisingly, the interaction occurred between SmMAPK3 and the entire JAZ family. Then, we further verified the interaction between SmMAPK3 and protein members in JA signalling pathways signals in tobacco leaves when nLUC-SmJAZ1 and nLUC-SmMYC2a were coexpressed with cLUC-SmMAPK3. Overall, these results demonstrated that SmMAPK3 interacts with SmJAZ2/3/4/5/6/8/9/ 10 in the JA signalling pathway.(SmJAZ1/2/3/4/5/6/8/9/10 and SmMYC2a) by performing an LCI assay. We used the empty vector with N-terminal domains and another empty vector with C-terminal domains together with every cLUC-fusion protein and nLUC-fusion protein construct as negative controls (Fig. 6B). As shown in Fig. 6C, strong fluorescent signals were detected when nLUC-SmJAZ2/3/4/5/6/8/9/10 was coexpressed with cLUC-SmMAPK 3 in tobacco leaves. However, we did not detect fluorescent 3. Discussion 3.1. SmMAPK3 is conserved and functions in phenolic acid accumulation Elicitor induction is considered an effective way to enhance the production of secondary metabolites. Research shows that SA, MeJA, H 2 O 2, SNPs and CaCl 2 promote the yields of phenolic acids (Ma et al., 2013). At the same time, SmMAPK3 responds to elicitors to different degrees (Fig. 3), indicating that SmMAPK3 likely participates in regulating the yields of phenolic acids. MAPK cascades have been recently reported to be involved in modulating plant secondary metabolism. ZmMPKL1 mediates the regulation of ABA accumulation in maize (Zhu et al., 2020), the OsMKKK10-OsMKK4-OsMPK6 cascade regulates cytokinin metabolism in rice (Guo et al., 2020), phosphorylation of the transcription factor AtWRKY33 regulates camalexin and indole glucosinolate biosynthesis via AtMPK3/ 6 in Arabidopsis (Yang et al., 2020; Zhou et al., 2020), AtMPK6-mediated phosphorylation of AtPIP5K6 inhibits the production of PtdIns (4,5)P 2 (Menzel et al., 2019), AtMPK4 interacts with AtMYB75 to increase the stability of AtMYB75 and the biosynthesis of anthocyanin via phosphorylation (Li et al., 2016), and CrMAPKKK1-CrMAPKK1-CrMAPK3/6 regulates the biosynthesis of the terpenoid indole alkaloid in Catharanthus roseus (Paul et al., 2017). Based on our observations, SmMAPK3 participates in regulating the accumulation of phenolic acids (Fig. 4). Gene expression of phenylpropanoid (SmPAL, SmC4H and Sm4CL), tyrosine (SmTAT and SmHPPR) and phenolic acid pathways (SmRAS and SmCYP98A14), which are necessary for the biosynthesis of phenolic acids, was significantly increased in OE plantlets (Fig. 4). 3.2. SmMAPKK2/4/5/7-SmMAPK3-SmJAZs is a newly identified pathway that regulates secondary metabolism It has been reported that the MKK4/5–MPK3/6 module is necessary for pathogen-induced malate metabolism (Su et al., 2017), primary root growth (Zhu et al., 2020) and auxin-facilitated lateral root emergence (Zhu et al., 2019) in Arabidopsis. SmMAPKK4, SmMAPKK5 and SmMAPKK7 are homologous genes of AtMKK4/5 and belong to group C (Xie et al., 2020). In stigmas, AtMKK2 is required to transmit upstream signals to AtMPK3 to mediate compatible pollination (Jamshed et al., 2020). SmMAPKK2 and SmMAPK3 are homologues of AtMKK2 and AtMPK3, respectively (Xie et al., 2020), and researchers have speculated that SmMAPKKKK3-Sm- MAPKKK83/59/41-SmMAPKK2-SmMAPK3 might be involved in phenolic acid biosynthesis. Therefore, SmMAPKK2, SmMAPKK4, SmMAPKK5, and SmMAPKK7 are probably upstream proteins of SmMAPK3. Fig.5 shows that SmMAPK3 physically interacts with SmMAPKK2/4/5/7. As a consequence, SmMAPKK2, SmMAPKK4, SmMAPKK5 and SmMAPKK7 are required to transmit upstream signals to SmMAPK3. We confirmed that SmMAPK3 controls the accumulation of phenolic acids in S. miltiorrhiza plantlets by regulating hormone signalling members (Fig. 7). Recent research shows that SmJAZ3 negatively regulates biosynthesis of the tanshinone pathway in S. miltiorrhiza through the SmWD40-170 protein, which is a positive regulator (Li et al., 2021b). SmJAZ8 plays a negative role in JA-induced biosynthesis of phenolic acids and tanshinones by interacting with SmMYC2a (Pei et al., 2018), and SmJAZ3 and SmJAZ9 act as repressors in tanshinone biosynthesis (Shi et al., 2016). Our results demonstrated that SmMAPK3 interacts with SmJAZ2/3/4/5/6/8/9/ 10 in the JA signalling pathway (Fig. 6A–C). Therefore, SmMAPK3 regulates the accumulation of secondary metabolites through SmJAZs in S. miltiorrhiza. However, interestingly, SmMAPK3 positively regulates the biosynthesis of phenolic acids. Recent research has reported that MdSnRK1.1 promotes the biosynthesis of proanthocyanidins and anthocyanins by phosphorylating the MdJAZ18 protein, which is a negative regulator (Liu et al., 2017). Phosphorylation of MdJAZ18 facilitates its 26S proteasome-mediated degradation, which releases MdbHLH3, thereby enhancing the expression of structural and regulatory genes (Liu et al., 2017). The phosphorylation of OsJAZ4 by OsGSK2 disrupts OsJAZ4-OsJAZ11 dimerization and the OsJAZ4-OsNINJA complex, leading to the degradation of OsJAZ4 and ultimately promoting plant antiviral defence (He et al., 2020). Therefore, we speculate that MAPK3 likely phosphorylates JAZ proteins and facilitates JAZ degradation. A previous study found that SmMAPK3 interacts with SmAREB1, SmMYB36/39/111 and SmPAP1 (Xie et al., 2020). SmAREB1, SmMYB111 and SmPAP1 act as positive regulators (Hao et al., 2016; Jia et al., 2017; Li et al.