Carbon dioxide sensing in Arabidopsis thaliana mediated by liquid-to-liquid phase separation of Protein Phosphatase Type 2C homologs

Abstract

Carbon dioxide sensing is crucial for plants to maintain a balance between exchange of gas and water vapor which directly affects tolerance of environmental stresses and ensures plant survival. Understanding CO2 sensing in plants will therefore help to be more prepared for future challenges in food crop production caused by increased environmental stresses due to climate change. An up to this point nearly unstudied process in plants, the phase separation (PS) of proteins, has recently been shown in vitro to be the main mechanism of CO2 sensing of a protein phosphatase type 2C (AP2C3) from Arabidopsis thaliana. PS describes the process of protein condensate droplet formation due to local enrichment of protein caused by interactive motifs usually located in an intrinsically disordered region (IDR) within the polypeptide backbone. In this study, we attempted to reproduce published results and screen for further IDR/PS mediated CO2 sensitivity of PP2Cs by cloning, expression, and purification of AP2C1, AP2C4 and PP2C74, along with AP2C3, for in vitro imaging experiments. We successfully established a reliable laser scanning confocal microscopy imaging setup for monitoring the phase separation of purified proteins fused to fluorescent tags. However, CO2-condensation of PP2C proteins could not be detected. Furthermore, we aimed to study PS and verify intracellular localisation of selected PP2C homologs in planta via a transient overexpression assay in Nicotiana benthamiana. Additionally, a connection of AP2C3 with the guard cell CO2 signalling pathway by a bimolecular fluorescence complementation assay with Mitogen-activated Protein Kinase 12 (MPK12) was investigated. These experiments verified the intracellular localisation of selected homologs, the presence of biomolecular condensates for AP2C3/4 in planta and indicated interaction between AP2C3 and MPK12.