The continuing rise in the atmospheric carbon dioxide (CO 2 ) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO 2 sensor remains unknown. Here, we show that elevated CO 2 triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO 2 , HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO 2 . Dominant HT1 mutants abrogate the CO 2 /bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO 2 sensor function and CO 2 -triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO 2 /bicarbonate sensor upstream of the CBC1 kinase in plants.

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Endophytic yeasts of genus Rhodotorula are gaining importance for their ability to improve plant growth. The nature of their interaction with plants, however, remains unknown. Rhodotorula mucilaginosa JGTA-S1 was isolated as an endophyte of Typha angustifolia and promoted growth in the host. To investigate the life-strategy of the yeast from a genomics perspective, we used Illumina and Oxford Nanopore reads to generate a high-quality annotated draft assembly of JGTA-S1 and compared its genome to three other Rhodotorula yeasts and the close relative Rhodosporidium toruloides . JGTA-S1 is a haploid yeast possessing several genes potentially facilitating its endophytic lifestyle such as those responsible for solubilizing phosphate and producing phytohormones. An intact mating-locus in JGTA-S1 raised the possibility of a yet unknown sexual reproductive cycle in Rhodotorula yeasts. Additionally, JGTA-S1 had functional anti-freezing genes and was also unique in lacking a functional nitrate-assimilation pathway—a feature that is associated with obligate biotrophs. Nitrogen-fixing endobacteria were found within JGTA-S1 that may circumvent this defective N-metabolism. JGTA-S1 genome data coupled with experimental evidence give us an insight into the nature of its beneficial interaction with plants.

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.

Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raflike kinase HT1 as a required step for stomatal movements caused by changes in CO 2 concentration. However, whether MPK12 kinase activity is required for regulation of CO 2 -induced stomatal responses warrants in-depth investigation.We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO 2 signaling that relies on allosteric inhibition of HT1.We show that CO 2 /HCO 3 À -enhanced MPK12 interaction with HT1 is independent of its kinase 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 CO 2 -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 CO 2 /HCO 3 À 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 guard cells sense changes in atmospheric CO 2 concentration.