Author: Rupinder Kaur Grewal
Grewal, Rupinder Kaur, 2025 Quantifying alternative oxidase proteins as biomarkers for environmental stress tolerance in wheat, Flinders University, College of Medicine and Public Health
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Triticum aestivum (wheat) is a globally important staple crop whose productivity is under the growing threat of environmental stressors including heat and drought. Plants employ a range of strategies to tolerate stress, including the alternative pathway of respiration, featuring the alternative oxidase (AOX) protein family. AOX prevents over-reduction of the mitochondrial electron transport chain, preventing the over-accumulation of reactive oxygen species (ROS) and protecting cellular homeostasis in stressful conditions. Understanding the expression and regulation of specific AOX isoforms in wheat during stress is therefore an important area of research. T. aestivum contains 13 AOX genes, distributed across the A, B and D subgenomes and classified into three subfamilies, -TaAOX1a, TaAOX1c, and TaAOX1d. TaAOX genes show unique expression patterns in response to stress, however there is currently no high-throughput method to quantify individual protein family members in wheat. New research aims to develop ELISAs to quantify TaAOX proteins at the sub-family level. At the start of this project, the immunisation of mice with an epitope from each sub-family had begun, but there was no full-length protein to test for sera reactivity nor validate ELISA protocols. Therefore, this project involved recombinant expression of TaAOX isoforms in a bacterial system, to test the reactivity of mouse sera. Sequence optimization of the AOX coding sequences was performed to maximise the efficiency of translation in Escherichia coli. The optimized sequences were supplied in pET22b (+) vector and expressed in BL21(DE3) cells. The recombinant protein expression was verified using SDS-PAGE and western blotting with a pan-specific AOX antibody. TaAOX1a and TaAOX1d were strongly expressed, however TaAOX1c expression required optimisation. The recombinant proteins were then used to screen sera from mice immunised with TaAOX subfamily-specific antigens. Sera were also tested against native AOX using mitochondria isolated from wheat seedlings. This study provides an advance in the development of tools for subfamily-specific detection of AOX protein in wheat including recombinant TaAOX proteins, future monoclonal antibodies and a platform for testing AOX ELISA protocols. Together, these tools will facilitate a new understanding of mitochondrial responses to stress in wheat and provide potential strategies to improve crop stress resilience through molecular breeding or biotechnology.
Keywords: drought, heat, AOX, Environmental stress, wheat
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Dr. Crystal Sweetman