The role of GABA in plant salinity and hypoxia responses

Abstract

My research is focused on establishing a better understanding of how γ-aminobutyric acid, GABA, contributes to the stress responses of plants, using the model plant Arabidopsis thaliana wild type (WT) and GABA mutants. Some mutants target decreased GABA production due to T-DNA insertion(s) in the major GABA producing gene(s) Glutamate Decarboxylase (GAD). Others target increased GABA poduction through overexpression of GAD or T-DNA knockout in the sole GABA catabolising gene GABA-transaminase (GABA-T). The two GAD1 mutants were genotyped and the GAD1 expression was examined in both the roots and shoots; while gad1KO was confirmed as a knock out line of GAD1, gad1* had high levels of expression of GAD1 in both shoots and roots that was comparable, if not greater than WT. Similarly, pop2-8, which cannot degrade GABA, had significantly higher GABA concentration in their leaves than all other lines; while the GAD2-overpression lines did not. In plants, the rapid accumulation of GABA in response to abiotic and biotic stress is common, including salinity and hypoxia. During my PhD I have induced salt and hypoxia stress to the above lines, explored their differences in response to salinity and submergence (hypoxia), and found that the mutants had varied tolerance to these two types of abiotic stress. Young and mature seedlings grown in square petri dishes and hydroponic system, respectively, were used to examine the impact of salt on root elongation, biomass and shoot ion content. To examine the response of plants with altered GABA metabolism to hypoxia, plants were grown for up to 4.5 weeks in short day conditions, then submerged in water for 6 d under light/dark cycles (slightly dimmed light during the day to represent the natural floods), and recovered for 2 d. This research highlights the importance of a tightly controlled and adjusted GABA content through GAD and GABA-T, as well as the essential role of GAD1 expression for plants in response to salinity and hypoxia. When GABA cannot be utilised (in the case of pop2-8 ), these plants had larger root growth and biomass compared to WT plants under normal and salt stress, but this appears to result in deficient recovery after submergence. After submergence, GAD1 expression was largely up-regulated in all plants with a higher fold change than that previously reported for GAD4. Moreover, GAD4 and GABA-T both impact the regulation of circadian rhythm, which were not significantly affected in the GAD1 lines under normal conditions. RNAseq and metabolite data suggests GABA regulation of hypoxia responses appears to occur through regulating circadian rhythm, amino acid metabolism, organic acid metabolism and transport, signalling transduction and photosynthesis. GABA biosynthesis (in the cytosol) consumes protons while the final step in catabolism (in the mitochondria) produces protons, thus GABA metabolism can alter pH in a cell. The final aim of this thesis was to examine how pH regulates the activity of Aluminum-activated Malate Transporter proteins (ALMTs), which are proteins shown to catalyse the movement of carboxylate ions across membranes and are inhibited by GABA. Specifically, the impact of a histidine residue within the putative GABA binding domain of wheat (Triticum aestivum, Ta) TaALMT1, was examined for its role in regulating the pH sensitivity of its malate transport capacity. TaALMT1 has greater malate-activated malate transport at alkaline pH than at acidic pH. The histidine residue is completely conserved among plant species, so its impact on transport of ALMT was studied here for the first time, in terms of its role in ion transport and pH sensitivity. Histidine carries a positive charge below pH 6 but is neutral above, therefore changes in pH would influence the charge states of histidine, and potentially its role in proton sensing and regulating activity of TaALMT1 at different pHs. This study conducted a single substitution of histidine to either alanine (H224A) or arginine (H224R) in TaALMT1, expressed the cRNA in Xenopus laevis oocytes, and then recorded the steady state currents in solutions of different pH (with or without malate). The results showed the H224R mutation increased, while the H224A inhibited, the external malate activated currents (mediated by TaALMT1) at both acidic (pH 4.5 and 5.5) and alkaline (pH 7.5) pH. These results indicate the importance of the H224 residue in maintaining the activity of TaALMT1 protein for ion transport but could not alter pH sensitivity of the protein. Taken together, this thesis mainly focused on the role of GABA metabolism under salinity and hypoxia, and also explored the role of histidine within the putative GABA binding motif in pH sensing for the pH sensitivity of ion transport. Knowledge from the current study has expanded our understanding of how GABA contributes to plant stress tolerance and signal transduction, and may be valuable for developing resilient crops given the fact that salinity and flooding are two of the major hazardous disasters happening not only in Australia, but also worldwide.