A proteomic approach to investigate the response of tef (Eragrostis tef) to drought

• Main Author: Kamies, Rizqah
• Other Authors: Rafudeen, Mohamed S
• Format: Doctoral Thesis
• Language: English
• Published: University of Cape Town 2016
• Subjects: Molecular and Cell Biology
• Online Access: http://hdl.handle.net/11427/16683

Includes bibliographical references === Eragrostis tef, commonly known as tef, is an important staple food and forage crop indigenous to Ethiopia. Tef plants are highly adaptable to abiotic stress conditions and are able to grow and produce grain yields under a wide range of environmental conditions, particularly under drought stress. In this study, tef plants were subjected to controlled dehydration stress treatment and physiologically characterised using relative water content (RWC), electrolyte leakage and chlorophyll fluorescence measurements, to establish critical water content stages for investigation of changes to the tef proteome in response to dehydration stress. Physiological testing showed tef viability to be retained to 30% RWC, however, further water loss to below 30% RWC, resulted in total loss of viability. Physiological characterisation with dehydration treatment showed a maximum leakage rate of 780 μS.min-¹.gdw-¹ and complete photosynthetic disruption with Fv/Fm and ɸPSII values decreasing to 0.2, below 30% RWC. Additionally, ultra-structural analysis using transmission electron microscopy showed extensive damage to the subcellular organisation of tef plant cells at water contents below 30% RWC. Based on these physiological data, it was decided to investigate the proteome of tef leaf dehydrated tissues at 50% RWC, as a non-lethal dehydration stress, as compared to hydrated tissues at 80% RWC. Proteomic analyses using iTRAQ mass spectrometry coupled to peptide OFFGEL fractionation and appropriate database searching with the Tef Extended and Liliopsida databases enabled the generation of three dataset results. These datasets, each contained a substantial amount of database matched proteins, where 5727 proteins for the Tef Extended (TE), 2656 proteins for the Tef Extended unique (TEU) and 4328 proteins for the Monocot unique (MU) datasets, were identified. Statistical analyses on peptide relative quantification values showed differential regulation of 211 proteins for the TE dataset, 111 proteins for the TEU dataset and 174 proteins for the MU dataset, in response to dehydration stress. A reciprocal BLAST search through the use of OrthoMCL with all three differentially regulated datasets (foregrounds) showed the TE foreground to provide the most comprehensive total protein coverage for further bioinformatics inference. Bioinformatics analysis using the programs Mercator, MapMan and Blast 2GO showed a diverse range of biological processes, where functional enrichment of GO-terms involved in biotic and abiotic stress response, signalling, transport, cellular homeostasis and pentose metabolic processes were enriched in tef high-abundance proteins. GO-terms linked to ROS producing processes such as photosynthetic reactions, cell wall catabolism, manganese transport and homeostasis, the synthesis of sugars and cell wall modification were enriched in tef low-abundance proteins. Additionally, KEGG analysis was used to observe tef proteins mapped to various biological pathways, of which the stress-responsive pathways, glutathione metabolism and ascorbate and aldarate metabolism were analysed in depth. Furthermore, biological validation of a few high-abundance proteins generated from iTRAQ analysis in the form of western blotting and relevant enzyme assays were conducted. The results showed the proteins fructose-bisphosphate aldolase (FBA), glutamine synthetase (GLN), functioning in plant maintenance 2 processes as well as the stress-protective antioxidant proteins, monodehydroascorbate reductase (MDHAR), peroxidase (POX) and superoxide dismutase (SOD) to be accumulated and further support iTRAQ findings. To date, this is the first study that has investigated the proteome profile of tef in response to dehydration.