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.
Highlights
Salvia miltiorrhiza Bunge is one of the traditional bulk medicinal materials
We aimed to identify the orthologous genes of sucrose non-fermenting-1-related protein kinase 2 (SnRK2) in S. miltiorrhiza, and to evaluate whether these proteins contribute to the regulation of phenolic acid accumulation and whether they are involved in the stress responses of this plant
These phylogenetic trees were constructed via MEGA7.0, using the neighbor-joining method with 1,000 bootstrap replicates. Since it overlapping with the plasma membrane marker, and in the cytoplasm and nucleus of the N. benthamiana epidermal cells. These results indicated that SmSnRK2.3 and SmSnRK2.6 were located in the cell membrane, cytoplasm, and nucleus, which are consistent with the subcellular localization of TaSnRK2.4/2.7/2.8, and ZmSAPK8 (Mao et al, 2010; Zhang H. et al, 2010; Ying et al, 2011; Zhang et al, 2011)
Summary
Salvia miltiorrhiza Bunge (family Labiatae) is one of the traditional bulk medicinal materials. Its dried roots have been prescribed for the clinical treatment of many human diseases, such as irregular menstruation, cardiovascular and cerebrovascular diseases, and inflammation (Kai et al, 2011; Ma et al, 2013). This plant contains two main bioactive ingredients: lipid-soluble tanshinones and water-soluble phenolic acids (Chen et al, 2001). Many studies have since focused on increasing this species phenolic acid contents by either overexpressing or suppressing its transcription factors or key enzyme genes within the relevant metabolic pathways. The overexpression of SmTAT, SmC4H, SmHPPR, AtPAP1, AtEDT1, and SmPAP1 induced a substantial accumulation of phenolic acids in the transgenic S. miltiorrhiza (Zhang Y. et al, 2010; Xiao et al, 2011; Hao et al, 2016; Liu Y. et al, 2016), whereas the downregulation of SmMYB39, SmHPPD, SmCCR1, or SmCHS all increased the content of phenolic acids (Xiao et al, 2011; Wang et al, 2012; Zhang et al, 2013; Zhang et al, 2015)
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