The Protein Kinase SmSnRK2.6 Positively Regulates Phenolic Acid Biosynthesis in Salvia miltiorrhiza by Interacting with SmAREB1

Abstract

Subclass III members of the sucrose non-fermenting-1-related protein kinase 2 (SnRK2) play essential roles in both the abscisic acid signaling and abiotic stress responses of plants by phosphorylating the downstream ABA-responsive element (ABRE)-binding proteins (AREB/ABFs). This comprehensive study investigated the function of new candidate genes, namely SmSnRK2.3, SmSnRK2.6, and SmAREB1, with a view to breeding novel varieties of Salvia miltiorrhiza with improved stress tolerance stresses and more content of bioactive ingredients. Exogenous ABA strongly induced the expression of these genes. PlantCARE predicted several hormones and stress response cis-elements in their promoters. SmSnRK2.6 and SmAREB1 showed the highest expression levels in the leaves of S. miltiorrhiza seedlings, while SmSnRK2.3 exhibited a steady expression in their roots, stems, and leaves. A subcellular localization assay revealed that both SmSnRK2.3 and SmSnRK2.6 were located in the cell membrane, cytoplasm, and nucleus, whereas SmAREB1 was exclusive to the nucleus. Overexpressing SmSnRK2.3 did not significantly promote the accumulation of rosmarinic acid (RA) and salvianolic acid B (Sal B) in the transgenic S. miltiorrhiza hairy roots. However, overexpressing SmSnRK2.6 and SmAREB1 increased the contents of RA and Sal B, and regulated the expression levels of structural genes participating in the phenolic acid-branched and side-branched pathways, including SmPAL1, SmC4H, Sm4CL1, SmTAT, SmHPPR, SmRAS, SmCHS, SmCCR, SmCOMT, and SmHPPD. Furthermore, SmSnRK2.3 and SmSnRK2.6 interacted physically with SmAREB1. In summary, our results indicate that SmSnRK2.6 is involved in stress responses and can regulate structural gene transcripts to promote greater metabolic flux to the phenolic acid-branched pathway, via its interaction with SmAREB1, a transcription factor. In this way, SmSnRK2.6 contributes to the positive regulation of phenolic acids in S. miltiorrhiza hairy roots.

Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism

“…5 a). SmAREB1 has been demonstrated to promote the flux of metabolites through the phenolic acid-branched pathway via its phosphorylation of SmSnRK2.6 [ 59 ]. The overexpression of SmMYC2 promotes the production of Sal B [ 60 ].…”

“…SmMAPK3 can physically interact with SmMYB36/SmMYB39/SmMYB111/SmPAP1/SmAREB1 (Fig. 8 ), which have been reported to regulate the synthesis and accumulation of secondary metabolites [ 57 , 59 , 67 – 69 ]. In A. thaliana , MAPKK9 promotes ethylene and camalexin biosynthesis [ 70 ], and both AtMPK3 and AtMPK6, which are highly involved in the plant response to biotic [ 71 – 75 ] and abiotic stress [ 21 , 54 , 76 ] and the regulation of ETH [ 17 , 26 , 33 ], SA [ 29 , 75 ] and JA [ 33 , 77 ] production, are activated by the upstream regulatory MAPK kinases AtMKK4 and AtMKK5 [ 78 , 79 ], which are in turn regulated by the upstream MAPKK kinase AtMEKK1 [ 80 ].…”

“…Gene-specific primers (Additional file 1 Table S7) were designed with Primer Premier v5.0 software to detect the expression of relevant genes. The expression levels of target genes were normalized to those of β-actin and ubiquitin [59].…”

The Ethylene Response factor SmERF1 Improves Salinity Tolerance and Impacts Seed Size in Tobacco

“…For example, genes of three multigene universal stress proteins (SmUSPs) were cloned, and their expression enhanced Escherichia coli tolerance to salt and heat stress [29]. Overexpression of SmSnRK2.6 ( sucrose non-fermenting-1-related protein kinase 2) improved S. miltiorrhiza tolerance to abiotic stresses [30]. Overexpression of SmLEA (late embryogenesis abundant proteins) improves drought and salinity tolerance in S. miltiorrhiza [31].…”

Salvia castanea Hairy Roots are More Tolerant to Phosphate Deficiency than Salvia miltiorrhiza Hairy Roots Based on the Secondary Metabolism and Antioxidant Defenses

“…Phenylalanine ammonia lyase (PAL) and tyrosine aminotransferase (TAT) are the rate-limiting enzymes in these pathways, respectively. Rosmarinic acid synthase (RAS) and cytochrome P450-dependent monooxygenase (CYP98AH14) are the key enzymes in the downstream of phenolic acid biosynthesis [ 18 ]. Tanshinones are mainly synthesized by the mevalonate (MVA) and 2-C-methyl- d -erythritol-4-phosphate (MEP) pathways.…”