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 <i>SmSnRK2.3</i>, <i>SmSnRK2.6</i>, and <i>SmAREB1</i>, with a view to breeding novel varieties of <i>Salvia miltiorrhiza</i> 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 <i>cis</i>-elements in their promoters. <i>SmSnRK2.6</i> and <i>SmAREB1</i> showed the highest expression levels in the leaves of <i>S. miltiorrhiza</i> seedlings, while <i>SmSnRK2.3</i> exhibited a steady expression in their roots, stems, and leaves. A subcellular localization assay revealed that both <i>SmSnRK2.3</i> and <i>SmSnRK2.6</i> were located in the cell membrane, cytoplasm, and nucleus, whereas <i>SmAREB1</i> was exclusive to the nucleus. Overexpressing <i>SmSnRK2.3</i> did not significantly promote the accumulation of rosmarinic acid (RA) and salvianolic acid B (Sal B) in the transgenic <i>S. miltiorrhiza</i> hairy roots. However, overexpressing <i>SmSnRK2.6</i> and <i>SmAREB1</i> 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 <i>SmPAL1</i>, <i>SmC4H</i>, <i>Sm4CL1</i>, <i>SmTAT</i>, <i>SmHPPR</i>, <i>SmRAS</i>, <i>SmCHS</i>, <i>SmCCR</i>, <i>SmCOMT</i>, and <i>SmHPPD</i>. Furthermore, SmSnRK2.3 and SmSnRK2.6 interacted physically with SmAREB1. In summary, our results indicate that <i>SmSnRK2.6</i> 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 <i>SmAREB1</i>, a transcription factor. In this way, <i>SmSnRK2.6</i> contributes to the positive regulation of phenolic acids in <i>S. miltiorrhiza</i> hairy roots.