The expression levels of enzyme genes involved in the phenolic acid biosynthesis pathway inside the roots (Figure 7A). Our qRT-PCR benefits indicated that the majority of these genes, which includes SmHPPR1, SmHPPR2, SmHPPR3, Sm4CL1, Sm4CL9, SmRAS2, SmRAS4, and SmCYP98A14, were significantly up-regulated (Figure 7B), especially the expression amount of Sm4CL9, which showed the largest fold transform in each and every OE line. two.six. SmSPL6 Binds Straight for the Promoter of SmCYP98A14 and Sm4CL9 It was reported that SPLs can regulate the expression of target genes by straight 5-HT3 Receptor Agonist web binding for the GTAC motif of target genes [19]. We located that the GTAC motif existed within the promoter regions of Sm4CL9 and SmCYP98A14 (Figure 8A). A yeast one-hybrid (Y1H) assay was performed to α9β1 custom synthesis examine the physical interactions between the SmSPL6 and also the promoter regions of Sm4CL9 and SmCYP98A14. Our outcomes indicated that SmSPL6 could bind to the promoter regions in the two genes (Figure 8B). Also, a dualluciferase transient transcriptional assay was performed to investigate irrespective of whether SmSPL6 may activate/regulate the expressions of SmCYP98A14 and Sm4CL9, using the outcomes indicating that it did (Figure 8D). These findings confirmed that SmSPL6 binds straight to and activates the promoters of SmCYP98A14 and Sm4CL9 to market the biosynthesis of RA and SalB.Int. J. Mol. Sci. 2021, 22,9 ofFigure 7. Expression changes of enzyme genes for the phenolic acid biosynthetic pathway within the SmSPL6-overexpressed (OE) transgenic lines. (A) Proposed biosynthetic pathway for phenolic acids (red indicates genes activated by SmSPL6). TAT, tyrosine aminotransferase; HPPR, hydroxyl phenylpyruvate reductase; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, hydroxycinnamate-CoA ligase; RAS, rosmarinic acid synthase; and CYP, cytochrome P450 enzymes. (B) Expression changes of enzyme genes for the tyrosine pathway, phenylpropanoid pathway, and specific phenolic acid pathway within the SmSPL6-OE lines. The expression level inside the manage was set to 1 (shown as red dotted lines). All data are the means of 3 biological replicates, with error bars indicating SD; represents a significant distinction at p 0.05 compared with all the handle.Figure 8. SmSPL6 binds for the promoter regions of Sm4CL9 and SmCYP98A14 and activates their expression. (A) GTAC motifs within the promoter regions of Sm4CL9 and SmCYP98A14. Red rectangles represent the GTAC motif. (B) Yeast one-hybrid detected interactions involving the SmSPL6 plus the promoters of Sm4CL9 and SmCYP98A14. The p53HIS2/pGADT7-p53 and p53HIS2/pGADT7 served as positive and adverse controls, respectively. (C) Schematic diagram of constructs employed in assays of transient transcriptional activity. (D) SmSPL6 activates the expression of Sm4CL9 and SmCYP98A14. Effector SmSPL6 was co-transformed with p4CL9-LUC/pCYP98A14-LUC reporters. All information would be the indicates of three biological replicates, with error bars indicating SD; represents a important difference at p 0.05 compared with the handle.Int. J. Mol. Sci. 2021, 22,10 of3. Discussion 3.1. Function of SmSPL6 in Phenolic Acid Biosynthesis Phenolic acids are an intense area of research inside the secondary metabolism of S. miltiorrhiza. Earlier reports have shown that several elicitors influence the production of phenolic acids [34]. These elicitors might be divided into two groups (biotic and abiotic), together with the former containing both pathogenic and plant cell elements [35,36], and the latter which includes Ag+ [37], MeJA [.