CO2Response Screen in GrassBrachypodiumReveals Key Role of a MAP-Kinase in CO2-Triggered Stomatal Closure

Abstract

AbstractPlants respond to increased CO2concentrations through rapid stomatal closure which can contribute to increased water use efficiency. Grasses display faster stomatal responses than eudicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO2signal transduction mutants in grasses has not been reported. The grass modelBrachypodium distachyonis closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO2control mechanisms of stomatal movements in grasses, we developed a forward genetics screen with an EMS-mutagenizedBrachypodium distachyonM5 generation population using infrared imaging to identify plants with altered canopy leaf temperature at elevated CO2. Among isolated mutants, a “chill1” mutant exhibited cooler leaf temperatures than wildtype Bd21-3 parent control plants after exposure to increased [CO2].chill1plants showed strongly impaired high CO2-induced stomatal closure, despite retaining a robust abscisic acid-induced stomatal closing response. Through bulked segregant whole-genome-sequencing analyses followed by analyses of further backcrossed F4 generation plants and generation and characterization of CRISPR-cas9 mutants,chill1was mapped to a protein kinase,BdMPK5. Thechill1mutation impaired BdMPK5 protein-mediated CO2/HCO3-sensingin vitro. Furthermore, AlphaFold2-directed structural modeling suggests that the identified BdMPK5-D90Nchill1mutant residue is located at the interface with the HT1 Raf-like kinase. BdMPK5 is a key signaling component involved in CO2-induced stomatal movements, potentially functioning as a component of the CO2sensor in grasses.