Metabolic and proteomic responses of sorghum cell cultures to polyethylene glycol-induced osmotic stress

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Mosea, Dipuo
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University of the Free State
Climate change, population growth, and the emerging water crisis are negatively affecting agricultural productivity and, thus, food security. Climate change results in drought, which is a major osmotic stress. Osmotic stress triggers the overproduction and accumulation of reactive oxygen species, which result in oxidative stress. Plants also respond to drought stress by using a wide range of biochemical and molecular mechanisms. Examples include osmotic adjustment, antioxidant defense systems, the production of phytohormones, and changes in gene and protein expression patterns. Most of the major cereal crops are drought sensitive. However, sorghum (Sorghum bicolor) is well-adapted to survive under hot and dry conditions. Sorghum is thus a potentially good model system among cereals in drought stress response studies. The study aimed to evaluate sorghum cell cultures' metabolic and proteomic responses to polyethylene glycol (PEG)-6000- induced osmotic stress. The viability of the white sorghum cell cultures was monitored using the MTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay after the cells were treated with 10 and 20% PEG osmotic stress. The levels of 16 metabolites and a sugar was analysed using hydrophilic interaction chromatography (HILIC) liquid chromatography-mass spectrometry (LC-MS) for control and 10 and 20% PEG-treated white sorghum cell suspension cultures. After 72 hours of 10 and 20% PEG-induced osmotic stress treatments, total soluble proteins (TSP) were extracted, and quantified using the Bradford assay and separated using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) to determine the quality of the extracts. An isobaric tag for relative and absolute quantitation (iTRAQ) analysis was conducted in the extracts followed by bioinformatics analysis of the positively identified osmotic stress-responsive proteins. The cell viability assays showed that both 10 and 20% PEG affected the metabolic activities of white sorghum cell cultures differently and, indeed, triggered osmotic stress, and the cell viability of the stressed samples declined relative to the control. In response to the imposed osmotic stress levels, the metabolic profiles showed a dramatic decline in six amino acids namely leucine (Leu), methionine (Met), phenylalanine (Phe), serine (Ser) threonine (Thr), and valine (Val). A total of 177 and 229 white sorghum cell-cultured total soluble proteins were identified for the 10 and 20% PEG treatment experiments, respectively. Of these identified proteins, 28 and 48 were responsive to the 10 and 20% PEG treatments, respectively. Additionally, the study identified responsive proteins such as germins, peroxidases, and histones as proteins of interest because they were either uniquely responsive to severe stress or commonly responsive to the imposed osmotic stress. The results obtained added to the knowledge that can be used in breeding programmes for the improvement of cereal crops that are susceptible to drought stress.
Dissertation (M.Sc. (Botany))--University of the Free State, 2023