Plants respond to increased CO2 concentrations through rapid stomatal closure which can contribute to increased water use efficiency. Grasses display faster stomatal responses than dicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO2 signaling mutants in grasses has not been reported. The grass model Brachypodium distachyon is closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO2 control mechanisms of stomatal movements in grasses, we developed a forward genetics screen on an EMS-mutagenized Brachypodium distachyon M5 population using infrared imaging to identify plants with altered canopy leaf temperature at elevated CO2. A chill1 mutant was isolated, exhibiting consistently cooler leaf temperatures than wildtype control plants after exposure to increased [CO2]. chill1 plants showed strongly impaired high CO2-induced stomatal closure, despite retaining a robust response to abscisic acid. Through bulked segregant whole-genome-sequencing analyses and generation and characterization of CRISPR-cas9 mutants, chill1 was mapped to a protein kinase, BdMPK5. The chill1 mutation impaired bdmpk5 protein-mediated CO2/HCO3- sensing in-vitro consistent with AlphaFold2-directed structural modeling. BdMPK5 is a key signaling component involved in CO2-induced stomatal movements, potentially functioning as a component of the CO2 sensor in grasses.

Forward Genetic Stomatal CO2 Response Screen in Grass Brachypodium distachyon Reveals Central Function of a MAP Kinase in Early Stomatal CO2 Signal Transduction