GABA regulation of gas exchange in barley (Hordeum vulgare)

Abstract

Stomatal guard cells are the primary gatekeepers for gas exchange between plants and the atmosphere, and therefore, modulate the rate of photosynthesis (i.e. plant energy production) and transpiration (i.e. plant water loss). It is well known that carbon gain and water loss through stomatal regulation is critical to plant growth and development, which is impacted by the diurnal cycle and stress. During stress, rapid accumulation of GABA (γ-aminobutyric acid) can occur through the GABA shunt, which bypasses two stress-inhibited reactions of the mitochondrial based TCA cycle. This observation makes GABA well known as a stress metabolite in plants. Far beyond this, evidence in the literature is emerging that GABA may act as a signal to impact stomatal regulation was demonstrated in multiple dicot plants to enhance plant water use efficiency and drought resilience. However, the interaction of GABA in regulating the stomata of cereal monocots such as barley, which provides a large proportion of food worldwide, has been less well explored. Considering the economic importance of barley in Australia and preventing yield loss from more frequent environmental challenges due to climate change, studying GABA signalling in barley stomatal regulation would be a meaningful topic to the agriculture industry. Here, it was found through physiological assays GABA inhibited light-induced stomatal opening of barley on both epidermal strips and reduced gas exchange in intact leaves. In contrast, GABA inhibition of dark-induced stomatal closure was only seen on epidermal peels. Like darkness-induced closure, GABA reduced the ABA sensitivity of stomatal response in epidermal strips but did not reduce water loss from leaves during steady state conditions. The inconsistent results between experimental systems suggests mesophyll cells may contributes to GABA regulation of stomatal pore movement. Transcriptionally, gene expression profiling was explored following manipulation of GABA. With the external application of GABA and ABA on barley guard cell samples, the expression pattern of differentially expressed genes (DE genes) were distinct between GABA and ABA treatment with distinct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways highlighted for the different treatments. Alternatively, in the leaf samples from GABA deficient Arabidopsis mutants, many stress defence signals related GO terms were commonly down-regulated across mutants due to the absence of GABA. The results indicated that GABA could interact with other signals transcriptionally, and suggests that it may contribute to stress defence. Interestingly, the convergence point for the defence-signalling network – the MAPK signalling pathway was shown as enriched regardless of GABA deficiency or external application. The MAPK signalling pathway could be a point of interaction of GABA with other signals including ABA. Stomatal assays, such as those reported above, represent a significant bottleneck in research pipelines, adding much time as a large repetitive workload for researchers. StomataAI (SAI) was developed as a reliable and user-friendly tool that is able to measure the stomatal pore aperture of the model plant Arabidopsis (dicot) and the crop plant barley (monocot) via the application of deep computer vision. The reliability of predicted measurements was examined with the designed Average-Human/Machine Test. Throughout the test, SAI was capable of producing measurements in line with human experts and reproducing conclusions of published datasets in a fraction of the time taken manually. Hence, SAI boosts the number of images that biologists could evaluate at one time to obtain more accurate measurements. Overall, this thesis illustrated that GABA is likely to act as a signal at both physiological and transcriptional level. SAI provide a reliable, efficient and high-throughput solution for stomatal pore measurement. The above biological outcomes contribute to further knowledge base on GABA regulation of stomata, particularly in the monocot barley. We highlight future areas that now can be explored including GABA dosedependency effects and GABA-ALMT interaction.