MPK12 in stomatal CO₂signaling: function beyond its kinase activity

Abstract

Protein phosphorylation by kinases is a major molecular switch mechanism involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf-like kinase HT1 as a required step for stomatal movements by changes in CO2 concentration. However, whether MPK12 kinase activity is required for regulation of CO2-induced stomatal responses warrants in depth investigation. We apply genetic, biochemical, and structural modeling approaches to examining the non-catalytic role of MPK12 in guard cell CO2 signaling that relies on allosteric inhibition of HT1. We show that CO2/HCO3-enhanced MPK12 interaction with HT1 is independent of its phosphor-transfer activity. By analyzing gas exchange of plant lines expressing various kinase dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO2-dependent stomatal responses rely on MPK12's ability to bind to HT1 but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2/HCO3 and present structural modeling that explains the MPK12:HT1 interaction interface. These data add to the model that MPK12 kinase-activity-independent interaction with HT1 functions as a molecular switch by which stomatal guard cells sense changes in atmospheric CO2 concentration.

Dynamic model of carbon dioxide-induced stomatal closure reveals a feedback core for cellular decision-making

“…This framework complements conventional biological approaches that often focus on single regulators or single pathways. Our analysis supports recent findings that HT1 is a key decisionmaking node of the CO2 signaling system [13,14], and offers additional insights regarding the ambient CO2 setting: closure induced by, e.g., exogenous ROS or Ca 2+ in ambient CO2 must involve the inactivation of HT1 and the activation of a positive feedback loop (the Main Stable Motif, formed by ROS, OST1 and the PP2Cs). Conversely, under conditions that activate HT1 the nodes of this positive feedback loop may also lock-in to their opposite states and lead to closure OFF.…”

“…As the follow-up analyses require that each edge of the network points to a node, during network construction we redirected each evidence of influence on an edge to an equivalent influence on the target of the edge [34]. MPKs are known to inhibit HT1 by a physical interaction induced by bicarbonate and independent of the MPKs' kinase activity [13,14], and also are necessary in Ca 2+ -induced SLAC1 activation [35].…”

“…Mitogen-activated protein kinases 4 and 12 (MPK4/12) bind to HT1 and inhibit its kinase activity [11, 12]. Bicarbonate enhances and stabilizes the binding of MPK4/12 to HT1 [13, 14]. Arabidopsis RESISTANT TO HIGH CO 2 (RHC1), a MATE-type transporter, may also link elevated CO 2 concentration to repression of HT1, although this has been debated [15, 16].…”

“…(A) The high CO2 signaling network with 47 nodes and 95 edges. (B) Acyclic graph representation of the network highlighting the in-component, the stronglyconnected-component and the out-component, forming a hierarchical structure of signal transduction; (C) Illustration of the multi-node representation of signaling elements that participate in multiple pathways and mechanisms, using MPKs as an example.MPKs are known to inhibit HT1 by a physical interaction induced by bicarbonate and independent of the MPKs' kinase activity[13,14], and also are necessary in Ca 2+ -induced SLAC1 activation[35].…”

Species‐specific stomatal ABA responses in juvenile ferns grown from spores

“…Most studies have found that ferns close their stomata in response to elevated CO 2 , although the response is lower in magnitude and slower than in angiosperms (Franks & Britton-Harper, 2016;Hõrak et al, 2017;Lima et al, 2019;K€ ubarsepp et al, 2020;Wu et al, 2020). No response has been observed in a few cases (Brodribb et al, 2009;Brodribb & McAdam, 2013) pathway in angiosperms (Merilo et al, 2013;Chater et al, 2015;Dittrich et al, 2019;Movahedi et al, 2021), and thus, possibly also in ferns, in the model angiosperm Arabidopsis thaliana, CO 2induced stomatal closure largely relies on the signalling module involving the HT1 and MPK12 kinases (Hashimoto et al, 2006;Hõrak et al, 2016;Jakobson et al, 2016;Takahashi et al, 2022;Yeh et al, 2023). An HT1 homologue is expressed in the stomatabearing sporophyte of the moss Physcomitrium patens (Chater et al, 2013;O'Donoghue et al, 2013), but whether its function is conserved from mosses to angiosperms is unclear (Harris et al, 2020).…”