Molecular changes of sorghum cell suspension Cultures in response to exogenous abscisic acid

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Muthego, Dakalo
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University of the Free State
Abiotic stresses reduce the growth and productivity of crops, thus threatening food security. It is therefore, important to develop crops that can withstand harsh environmental conditions in order to ensure availability of food. In general, plants have developed a wide range of mechanisms in response to these abiotic stresses. For example, under stress conditions, plants undergo molecular changes which include alterations in gene, protein and metabolite expression patterns that are mostly regulated by the plant hormone abscisic acid (ABA). ABAregulated stress responsive pathways are well studied in the model plant Arabidopsis (Arabidopsis thaliana), yet similar processes in sorghum (Sorghum bicolor), a drought tolerant crop, are not yet fully understood. The aim of the study was to investigate the biochemical properties and protein expression patterns of sorghum cell suspension cultures in response to exogenous ABA. White sorghum cell suspension cultures were used, and at eight days postsubculture, the cultures were treated with 100 μM ABA prepared in 70% (v/v) methanol. For control cells, an equal volume of 70% (v/v) methanol was added, and both treatment groups were incubated with shaking at 27℃ for 72 hours. Analysis of cell viability using the 3-(4,5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that ABA does not affect the viability of the cells at 0, 24, 48 and 72 hours. However, exogenous application of ABA for 72 hours resulted in increased accumulation of proline in the sorghum cells relative to the controls. Furthermore, proteins were extracted from the cells, as total soluble proteins (TSP), and from the culture medium, as culture filtrate proteins (CF) 72 hours after the exogenous ABA treatment. The protein profiles of the two proteomes were visually analysed on Coomassie brilliant blue-stained one-dimensional sodium dodecyl sulfate polyacrylamide gels. The gels showed that the two proteomes were of good quality even under control conditions. Furthermore, following the 72-hour ABA treatment, proteins were differentially expressed in both the TSP and CF proteomes. Moreover, isobaric tags for relative and absolute quantitation (iTRAQ) method and mass spectrometry were used to identify and quantify the differentially expressed proteins. A total of 725 and 256 proteins were identified in the TSP and CF proteomes, respectively. Of all these, 46 and 82 were ABA-responsive in the TSP and CF, respectively, and 8 proteins were common to both proteomes. Signal peptide analysis revealed that the majority of TSP found in the intracellular matrix did not have a predicted signal peptide (72%), while the majority of CF proteins found in the extracellular matrix contained signal-peptides (82%). Amongst these differentially expressed proteins in both the TSP and CF proteomes, the majority of them proteins were involved in metabolism with 37% and 35%, followed by defence with 24% and 24%, respectively. However, the metabolic processes in the CF were mainly related to carbohydrate metabolism. The signal transduction functional group was only unique to the TSP fraction, while transporters, and cell structure functional groups were unique to the CF protein fraction. The differentially expressed proteins are well-known stress proteins such as peroxidases and superoxide dismutases whose levels change under abiotic stresses. Together with causing an increase in proline content, a known osmoprotectant, exogenous ABA does indeed act as a stress phytohormone. Furthermore, these results showed that ABA influences the differential expression of both intracellular and extracellular matrix proteins, possibly suggesting the importance of both cell compartments in stress response. Furthermore, these two compartments have different roles in stress responses as suggested by the results. Therefore, the application of exogenous ABA could be the way forward to further understand plant stress response networks, and possibly to develop crops that can survive under any abiotic stress.
Dissertation (M.Sc.(Plant Sciences))--University of the Free State, 2022
Sorghum, cell suspension cultures, exogenous abscisic acid, total soluble proteins, culture filtrate proteins, proline, signal peptides, intracellular proteins, extracellular proteins, iTRAQ