Casein kinase 2 α and ß subunits inversely modulate ABA signal output in Arabidopsis protoplasts.

MAIN CONCLUSION: Our transient gene expression analyses in Arabidopsis protoplasts support the view that CK2αs and CK2ßs positively and negatively modulate ABRE-dependent gene expression, respectively. The phytohormone abscisic acid (ABA) regulates the expression of thousands of genes via ABA-responsive elements (ABREs), and has a crucial role in abiotic stress response. Casein kinase II (CK2), a conserved Ser/Thr protein kinase in eukaryotes, is essential for plant viability. Although the CK2 has been known as a tetrameric holoenzyme comprised of two catalytic α and two regulatory ß subunits, each of the two types of subunits has been proposed to have independent functions. The Arabidopsis genome encodes four α subunits (CK2α1, CK2α2, CK2α3, CK2α4) and four ß subunits (CK2ß1, CK2ß2, CK2ß3, CK2ß4). There is a growing body of evidence linking CK2 to ABA signaling and abiotic stress responses. However, the roles of each CK2 subunit in ABA signaling remain largely elusive. Using the transient expression system with the core ABA signaling components in Arabidopsis leaf mesophyll protoplasts, we show here that CK2α1 and CK2α2 (CK2α1/2) positively modulate ABRE-dependent gene expression as ABA signal output in ABA signaling, whereas all four CK2ßs negatively modulate the ABRE-dependent gene expression mediated by subclass III SnRK2-AREB/ABF pathway and by CK2α1/2. These data indicate that CK2α1/2 and CK2ßs positively and negatively modulate ABA signal output, respectively, suggesting that the quantitative balance of CK2 subunits determines the ABA signal output in plants. Given that CK2s act as pleiotropic enzymes involved in multiple developmental and stress-responsive processes, our findings suggest that CK2 subunits may be involved in integration and coordination of ABA-dependent and -independent signaling.

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

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.