Exploring the Role of GABA in Stomatal CO2 Responses and Carbon Metabolism

Abstract

Rapid acclimation to alterations in environmental conditions is vital for the survival of plants in an everchanging world. This includes the adjustment of stomatal apertures in accordance with environmental stimuli as well as metabolic changes, particularly, those related to cellular respiration and photosynthesis. A signal that is synthesised rapidly and in large concentrations to a great number of different environmental factors is γ-aminobutyric acid (GABA). Recently, GABA was found to fine-tune stomatal opening by negatively regulating the anion channel Aluminium-activated malate transporter 9 (ALMT9) in response to stomatal opening stimuli. However, the GABA signalling pathway is largely unexplored. In this thesis, a potential link between GABA and high/low CO2 signalling in guard cells was investigated using mutant lines altered in their GABA metabolism. These lines were mutated in one or several genes encoding members of a family of GABA synthesis enzymes, known as Glutamate decarboxylases (GADs). Gas exchange measurements in corresponding GAD knockout plants revealed a wild-type stomatal response to low/high atmospheric CO2, except for one mutant line (gad2-1). Whole genome sequencing unveiled a second-site mutation in gad2-1 in a gene encoding a crucial regulator of high CO2 signalling, known as Mitogen‐activated protein kinase 12 (MPK12), as well as in its neighbour gene BYPASS2 (BPS2). This deletion mutation is associated with more open stomata and reduced CO2 sensitivity. Re-expressing MPK12 in the guard cells of gad2-1 restored CO2 sensitivity and proved that the aberrant CO2 phenotype of this mutant line was linked to the absence of MPK12 rather than the mutated GAD2. In this way, the study demonstrated that GABA signalling and the high/low CO2-induced signal transduction pathway do not overlap. Moreover, it was also shown that stomatal opening responses to low atmospheric CO2 occur independently of the opening channel ALMT9. Since GABA production is associated with the release of CO2 as a by-product into the cytosol, the impact of disrupted GABA synthesis on photosynthetic CO2 assimilation and biomass accumulation under ambient and high CO2 conditions was investigated in a different experimental series. The study provided data-driven evidence that GABA deficiency has no effect on the photosynthetic capacity of plants as well as on plant growth, neither under standard conditions nor upon high CO2 exposure. Instead, an RNA-seq analysis uncovered the upregulation of genes that are associated with carbohydrate and cell-wall metabolic processes. This was accompanied by largely elevated concentrations of the cell-wall monosaccharide xylose in the GABA-depleted mutant lines. Together, the presented findings here reject the hypothesis that GABA is involved in CO2 signalling in guard cells, as well as the speculation that GABA production affects CO2 uptake for photosynthetic carbon assimilation. Furthermore, this work provides detailed information about an unrelated mutation to the gene of interest in a commonly used gad knockout line to prevent misinterpretations of its stomatal phenotype in future studies. In addition, some novel, GABA-unrelated insights into guard cell function, which involve, inter alia, stomatal opening anion channels and their role in low CO2 responses, as well as some new aspects regarding GABA functioning in carbohydrate metabolism are discussed, thereby providing a platform for more in-depth research.