Identification, classification of AOX genes in T. aestivum and their expression patterns to salt stress

Author: Badr Alharthi

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Alharthi, Badr, 2020 Identification, classification of AOX genes in T. aestivum and their expression patterns to salt stress, Flinders University, College of Science and Engineering

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Abstract

Crop yields are subject to environmental stresses. Increasing the knowledge of stress-tolerant plant genotypes could increase crop yields. AOX genes have been shown to play a significant role in stress tolerance of several plant species. For crop species to date, research has focused on AOX in rice and barley and its stress responses, and it has been observed that AOX expression responds to low temperatures and salinity stress, as environmental stressors.

At the start of this project, publications on AOX and the alternative pathway of respiration in wheat were scarce. The primary objective of this research was to identify and characterise the AOX gene family in T. aestivum. Specifically, the aims were to (1) identify the gene candidates in commercial bread wheat; (2) assess which of these genes were stress responsive when exposed to chemical and a biological stress, such as salinity; and (3) assess AOX gene families’ members and expression to determine differences, if any, between T. aestivum and its wild ancestors during its evolution.

The research characterised the structure, phylogeny and expression profile of the AOX gene family. In T. aestivum and its related species, 89 AOX1 genes were identified, of which, 58 were confirmed as complete genes. However, owing to the quality of genome assemblies of Ae. sharonensis, it was not possible to determine the full length of AeshAox1a. From the phylogenetic analyses, those identified AOX proteins were classified as AOX1a, AOX1c, AOX1d and AOX1e type. The in silico analyses showed that the AOX genes in T. aestivum were expressed at precise developmental stages as well as when subjected to both abiotic and biotic stresses.

Four commercially significant T. aestivum cultivars, Chinese Spring (CS), Opata M85 (Op.), Gladius (Gl.) and Drysdale (Dr.), showed different salinity tolerances in response to salt stress. A comparative study between tolerant versus sensitive cultivars revealed tissue-dependent variations in AOX gene expression, physiological and biochemical responses. Thirteen Aox1 genes were studied at the transcript level using qRT-PCR. Under chemical and salinity stress, qRT-PCR analyses showed that Aox1a clades and Aox1d clades were the most responsive isoforms in T. aestivum.

Similarly, Aox1a clades and Aox1d clades genes were the most responsive Aox1 gene expression in four direct ancestors of T. aestivum. Consistent with the transcript findings, immunoblot analysis revealed that AOX protein abundance was induced by KCN and salinity stress. This study’s findings showed that the AOX protein abundance was higher in the seedling under KCN treatment than in the seedling under salinity stress. The findings suggest that the elevated AOX expression in both sensitive species reflects the role of AOX attempting to minimise ROS when subjected to salinity stress. In contrast, tolerant species exhibited higher pre-existing AOX protein levels than salt-sensitive species, which gives salt-tolerant species an advantage in coping with oxidative stress.

Keywords: alternative oxidase AOX, salinity tolerance, abiotic stress, gene overexpression, oxidative stress, Wheat, T. aestivum

Subject: Biotechnology thesis

Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Science and Engineering
Supervisor: Kathleen Soole