Constitutively active B2 Raf-like kinases are required for drought- responsive gene expression upstream of ABA- activated SnRK2 kinases

Osmotic stresses, such as drought and high salinity, adversely affect plant growth and productivity. The phytohormone abscisic acid (ABA) accumulates in response to osmotic stress and enhances stress tolerance in plants by triggering multiple physiological responses through ABA signaling. Subclass III SNF1-related protein kinases 2 (SnRK2s) are key regulators of ABA signaling. Although SnRK2s have long been considered to be self-activated by autophosphorylation after release from PP2C-mediated inhibition, they were recently revealed to be activated by two independent subfamilies of group B Raf- like kinases, B2- RAFs and B3- RAFs, under osmotic stress conditions. However, the relationship between SnRK2 phosphorylation by these RAFs and SnRK2 auto phosphor ylation and the individual physiological roles of each RAF subfamily remain unknown. In this study, we indicated that B2- RAFs are constantly active and activate SnRK2s when released from PP2C-mediated inhibition by ABA-binding ABA receptors, whereas B3- RAFs are activated only under stress conditions in an ABA-independent manner and enhance SnRK2 activity. Autophosphorylation of subclass III SnRK2s is not sufficient for ABA responses, and B2- RAFs are needed to activate SnRK2s in an ABA-dependent manner. Using plants grown in soil, we found that B2- RAFs regulate subclass III SnRK2s at the early stage of drought stress, whereas B3- RAFs regulate SnRK2s at the later stage. Thus, B2- RAFs are essential kinases for the activation of subclass III SnRK2s in response to ABA under mild osmotic stress conditions, and B3- RAFs function as enhancers of SnRK2 activity under severe stress conditions.