Exploring interactions of GABA and alternative oxidase pathways under salinity stress

Author: Madhuka Hansamali Kanapaddalagamage

Kanapaddalagamage, Madhuka Hansamali, 2022 Exploring interactions of GABA and alternative oxidase pathways under salinity stress, Flinders University, College of Medicine and Public Health

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Dryland salinity effects ~2.5 million hectares of Australian farmland and is expected to increase to 17 million hectares by 2050 leading to a loss in income of $270 million annually. Salinity affects growth, development and crop yield and poses a challenge to global food security. Plants growing in saline conditions show ionic imbalance, hyperosmotic stress, increased accumulation of gamma aminobutyric acid (GABA) and reactive oxygen species (ROS) leading to oxidative stress.

Studies suggest that non-phosphorylating alternative respiratory pathways mediated by NAD(P)H Dehydrogenase and Alternative Oxidase (AOX) play an important role during stress by uncoupling carbon metabolism from adenylate control and minimization of ROS. Further, increase in GABA, a non-protein amino acid helps plants cope with salinity induced osmotic stress by maintaining a balance between accumulation of sodium (Na+) and loss of potassium (K+).

This study investigated the interaction between GABA and AOX pathway under salinity stress. This first aim was addressed by exploring the effects of AOX overexpression on endogenous GABA concentration, expression of GABA-related (GAD2, GAD1) and AOX pathway related (AOX1a, AOX1d, NDB2) genes and levels of a marker (MDA) for reactive oxygen species, in the presence and absence of salinity stress. Arabidopsis thaliana wild type (Col0) and AOX1a overexpression line were grown in hydroponic system for 6 weeks and were treated with 100 mM NaCl for 1 and 9 days. Shoots and roots samples were harvested 1 day and 9 days after salt treatment. Transcript levels of AOX1a, AOX1d, NDB2 and GAD2 (relative gene expression for all the genes were obtained by 2- ΔΔCT method) , MDA content and endogenous GABA content in both the wild type and AOX1a overexpression line were increased under salinity stress. GABA content was higher in both shoot and root tissues of the AOX1A overexpression line relative to wild type, under control conditions.

The second aim of this study was to investigate the combined roles of AOX and GABA on salinity tolerance in Arabidopsis thaliana plants that were either overexpressing, or lacking, AOX1a. This


aim was addressed by exploring the effects of exogenous GABA application on plant physiology measurements, including shoot and root growth, stomatal conductance and ion accumulation in the presence and absence of salinity stress. Six-week-old wild type, AOX1a overexpression and AOX1a knock-out plants grown in soil were treated with sodium chloride (NaCl, 150 mM) with or without GABA (10 mM) for 10 days and harvested to measure fresh weight, dry weight, and sodium and potassium ion concentrations. Seedlings were also transferred to agar plates containing different concentrations of NaCl and GABA, for the measurement of total root length and relative growth rate.

Stomatal conductance was greatly reduced by salinity stress independent of the presence of exogenous GABA. However, sodium ion concentration, which can be toxic to the cell during salinity stress, was significantly lower in AOX1a knock-out plants if they were treated with GABA simultaneous to the salt treatment. Relative growth rate of roots in wild type and knock-out lines were reduced significantly when GABA was applied in the absence of salinity stress. However, under salinity stress conditions this negative growth effect was gone, and GABA improved the relative growth rate of AOX1a overexpressing roots, relative to wild type roots.

According to these results, there is some interplay between AOX and GABA, based on the increase in endogenous GABA levels in the AOX1a overexpression line, as well as decreased sodium ion concentrations and increased plant root growth in AOX1A knock-out lines treated with GABA. The AOX pathway may facilitate GABA metabolism, which in turn could act as a carbon or nitrogen source during stress, or there might be a common signaling pathway for both AOX and GABA metabolism. However, more work is required to support these findings and determine whether there is a direct interaction between the AOX and GABA shunt pathways.

Keywords: GABA, AOX, salinity stress

Subject: Biotechnology thesis

Thesis type: Masters
Completed: 2022
School: College of Medicine and Public Health
Supervisor: Sunita Ramesh