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

Abstract

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 autophosphorylation 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.

Plants lacking OST1 show conditional stomatal closure and wildtype‐like growth sensitivity at high VPD

“…There is still no answer to why OST1 loss‐of‐function plants generally show strong stomatal insensitivities to different abiotic stimuli, while there are many alternative kinases (CPK3, CPK6, CPK21, and CPK23) activating SLAC1 and other anion channels present (Merilo et al, 2013). Recently, B2 Raf‐like kinases were found to activate SnRK2s in an ABA‐dependent way under mild drought stress, whereas B3 Raf‐like kinases enhanced SnRK2 activity ABA‐independently under severe drought stress (Soma et al 2023). These regulations could also be involved in the active response of ost1‐3 , either via changes in hydraulic conductance or activation of stress‐induced gene expression.…”

Integrative regulatory mechanisms of stomatal movements under changing climate

“…However, in the presence of ABA, ABA receptors PYR1/PYLs /RCARs bind ABA to form a complex with PP2Cs and inhibit the activities of these PP2Cs (Ma et al, 2009;Park et al, 2009;Tischer et al, 2017). The released SnRK2s can be phosphorylated and activated by other protein kinases such as Rapidly Accelerated Fibrosarcoma-LIKE MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASEs (Raf-like MAPKKKs) (Saruhashi et al, 2015;Fàbregas et al, 2020;Lin et al, 2020;Soma et al, 2020Soma et al, , 2023Takahashi et al, 2020; Figure 1). Furthermore, activated-SnRK2s, such as SnRK2.6/OST1, positively regulate the activity of guard cell plasma membrane-localized R-type (QUAC1/ALMT12) and S-type (SLAC1) anion channels, thus closing the stomata (Geiger et al, 2009;Meyer et al, 2010;Sasaki et al, 2010;Imes et al, 2013).…”

“…These results indicate that additional proteins are required to initiate SnRK2.6/OST1 activity in planta. Recently, with a combination of forward genetic screen and phosphoproteomics approaches, several independent groups have reported and identified that MAPKKK-RAFs play an essential role in the activation of SnRK2s in Arabidopsis (Nguyen et al, 2019;Katsuta et al, 2020;Lin et al, 2020;Soma et al, 2020Soma et al, , 2023Takahashi et al, 2020;Lin et al, 2021;Wang et al, 2023a; Figure 1). Arabidopsis SnRK2s can be quickly activated in response to osmotic stress, which is independent of the ABA signaling pathway (Boudsocq et al, 2004(Boudsocq et al, , 2007Yoshida et al, 2002Yoshida et al, , 2006.…”

“…Arabidopsis SnRK2s can be quickly activated in response to osmotic stress, which is independent of the ABA signaling pathway (Boudsocq et al, 2004(Boudsocq et al, , 2007Yoshida et al, 2002Yoshida et al, , 2006. Interestingly, recent studies have found that drought stress can activate MAPKKK-B3-RAF3/4/5 in an ABA-independent way, and plays critical roles in activating SnRK2.2, SnRK2.3, and SnRK2.6/OST1 to cope with severe stress conditions (Lin et al, 2020;Soma et al, 2020Soma et al, , 2023Takahashi et al, 2020). The results showed that the MAPKKK-B3-RAF3/4/5 protein kinases are required to reconstitute ABA activation of SnRK2.6/OST1 in vitro and to activate SLAC1 by SnRK2.6/ OST1 in oocytes without split YFP fusion (Takahashi et al, 2020).…”

“…The results showed that the MAPKKK-B3-RAF3/4/5 protein kinases are required to reconstitute ABA activation of SnRK2.6/OST1 in vitro and to activate SLAC1 by SnRK2.6/ OST1 in oocytes without split YFP fusion (Takahashi et al, 2020). Moreover, under mild drought conditions, autophosphorylation of released SnRK2s is not sufficient to activate itself in response to ABA, and phosphorylation of SnRK2s by constitutively active MAPKKK-B2-RAF7/10/11/12 is required to activate them to regulate drought tolerance (Soma et al, 2023). Abscisic acid does not activate B2 or B3 RAF kinases (Lin et al, 2020;Soma et al, 2023), and SnRK2s are not activated by ABA in the raf7/10/11/12 quadruple mutant, suggesting that SnRK2s released after ABA treatment can be phosphorylated and activated by the constitutively active MAPKKK-B2-RAF7/10/11/12 (Soma et al, 2023).…”