Doctoral Degrees (Plant Sciences)

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Browsing Doctoral Degrees (Plant Sciences) by Advisor "Chivasa, Stephen"

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    Characterisation of the physiological, biochemical and molecular responses of sorghum to drought stress
    (University of the Free State, 2018-11) Goche, Tatenda; Ngara, Rudo; Chivasa, Stephen
    Drought is a major threat to global food security due to its detrimental effects on plant growth, productivity and yield quality. Many climatic models are predicting the increasing duration and severity of drought episodes. Therefore, understanding plant adaptive responses to drought stress is important in developing new biotechnological solutions to avert crop yield losses to drought. Sorghum (Sorghum bicolor) is an African indigenous crop that is well-adapted to thrive on marginal lands. This makes the crop a suitable model plant for studying adaptive responses to drought. In this study, the physiological and biochemical responses of two sorghum varieties with contrasting phenotypic traits to drought stress was analysed under drought stress. The two sorghum varieties used were the drought susceptible ICSB 338 and the drought tolerant SA 1441. The sorghum plants were grown in soil until the V3 growth stage before withholding water for 8 days, re-watering and then assessing the physiological changes following the drought stress treatment. Physiological analyses of the plants revealed striking differences between the sorghum varieties. The growth parameters of both roots and shoots exhibited more tolerance related responses in the drought-tolerant variety, while the susceptible variety was adversely affected and had poor recovery after re- watering. The leaf relative water content, stomatal conductance and chlorophyll content, supported the observed physiological adaptations. The analysis of proline and glycine betaine showed that there was an increase in the accumulation of the two omolytes in response to drought stress. The drought tolerant variety showed significantly higher osmolyte accumulation earlier than the drought susceptible variety in both root and shoot tissue. The isobaric tags for relative and absolute quantitation (iTRAQ) analysis was used to identify drought-stress responsive root proteins in the two sorghum varieties. In the root proteome, 1169 and 1043 proteins were positively identified for ICSB 338 and SA 1441 sorghum varieties, respectively. Of these proteins, 237 and 184 were drought responsive for ICSB 338 and SA 1441, respectively. A large proportion of the proteins are involved in disease/defence (26% for ICSB 338 and 23% for SA 1441) followed by metabolism (25% for ICSB 338 and 21% for SA 1441). To validate gene function, eight proteins with the highest fold-change in response to drought were selected for gene expression analysis using quantitative real time- polymerase chain reaction (qRT-PCR). The results showed that all the genes evaluated were drought stress responsive. In order to develop the eight target genes as drought markers, their expression was analysed in cell suspension cultures of White sorghum and ICSB 338 with and without sorbitol treatment. The gene expression analysis showed that seven of the eight drought responsive genes could distinguish between White sorghum and ICSB 338 in the cell suspension culture system without sorbitol treatment. In addition, all the eight genes could distinguish between White sorghum and ICSB 338 in response to the sorbitol-induced osmotic stress. Following this, the responses of the genes to heat stress was analysed in the White sorghum cell suspension cultures. The results showed that seven of the genes were also heat responsive. These genes are recommended for use as drought markers in marker assisted selection for drought tolerance. As proof-of-concept and to develop a workflow for the use of the drought markers in other crops, three published genes from our research group were used. Four Arabidopsis (Arabidopsis thaliana) homologues of each sorghum gene were selected for gene expression analysis. The results showed that there is differential gene expression between homologues of the same gene in response to osmotic stress. In conclusion, the comparative sorghum physiological, biochemical, protein and gene expression data generated in this study forms a foundation for further sorghum molecular studies. Furthermore, the drought marker genes toolkit developed in this study can be utilised by plant breeders in marker assisted selection for the improvement of agriculturally important crops against drought.
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    Physiological, biochemical and molecular analyses of the drought stress responses of two contrasting wheat varieties
    (University of the Free State, 2024) Moloi, Sellwane Jeanette; Ngara, Rudo; Chivasa, Stephen
    Wheat (𝘛𝘳𝘪𝘵𝘪𝘤𝘶𝘮 𝘢𝘦𝘴𝘵𝘪𝘷𝘶𝘮) is the second most widely grown cereal crop worldwide. It is primarily used for human consumption, animal feed, and industrial biofuels. However, the production of wheat is negatively affected by drought, and current climate models are predicting more frequent and severe drought episodes in the future. As such, agricultural productivity will be negatively affected. Therefore, understanding plant responses among plant varieties with different drought phenotypes could help identify traits related to drought tolerance and aid in developing more drought-tolerant crops. This study compared the physiological, biochemical, and molecular responses of the drought-tolerant (BW35695) and drought-susceptible (BW4074) wheat varieties to drought stress. The wheat plants were grown in potting soil for two weeks in a growth chamber before withholding water for 28 days. Drought stress significantly decreased soil moisture content in the water-deprived pots of both varieties relative to the well-watered controls. However, there was no significant difference in soil moisture content between the varieties, suggesting comparable levels of water deficit stress. Physiological and biochemical parameters such as leaf chlorophyll, carotenoid and relative water content (RWC), lipid peroxidation, reactive oxygen species (ROS), osmolyte content, and enzymatic antioxidant activities revealed striking differences between the varieties. The drought-tolerant wheat variety, BW35695, demonstrated remarkable resilience to the imposed drought stress by exhibiting higher leaf RWC, chlorophyll, carotenoid, and osmolyte content compared to the drought-susceptible variety, BW4074. Additionally, BW35695 mitigated drought-induced oxidative stress by enhancing the activities of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) in both leaves and roots and reduced ROS accumulation and membrane lipid damage. The leaf proteome of the wheat varieties was analysed to identify drought-responsive leaf proteins using the isobaric tags for absolute and relative quantitation (iTRAQ) method coupled with mass spectrometry. A total of 1062 and 882 leaf proteins were positively identified in BW4074 and BW35695 wheat varieties, respectively, of which 69 and 110 were drought responsive. Most of the drought-responsive leaf proteins in BW35695 were involved in energy (28%) and protein synthesis/folding/degradation (25%). For BW4074, primary metabolism (23%), energy (23%) and protein synthesis/folding/degradation (20%) were the most represented protein functional groups. The rest of the drought-responsive leaf proteins had putative functions in defence/ROS detoxification (20% and 10%), transcription (4% and 7%), secondary metabolism (6% and 3%), and cell structure (2% and 1%) in BW4074 and BW35695, respectively. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the two most significantly enriched pathways in BW4074 were alanine aspartate and glutamate metabolism, and arginine biosynthesis, while photosynthesis-antennae and photosynthesis were most enriched in BW35695. The KEGG pathway enrichment analysis supported the putative functional groupings data, which suggests that drought stress affects various biological processes in wheat leaves, particularly primary metabolism in BW4074 and photosynthesis in BW35695. Protein-protein interactions of the drought-responsive proteins were analysed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The results showed that primary metabolism and protein synthesis were the main interacting functional groups for BW4074, while photosynthesis and protein synthesis were prominently interacting in BW35695. Photosynthesis-related proteins were significantly suppressed in BW35695, possibly as a strategy to reduce ROS-induced cell damage, while protein synthesis-related proteins were increased, possibly to enhance the biosynthesis of other stress-responsive proteins. Six drought-responsive proteins were selected from the iTRAQ data for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The gene expression results revealed that a 𝘥𝘦𝘭𝘵𝘢-1-𝘱𝘺𝘳𝘳𝘰𝘭𝘪𝘯𝘦-5-𝘤𝘢𝘳𝘣𝘰𝘹𝘺𝘭𝘢𝘵𝘦 𝘴𝘺𝘯𝘵𝘩𝘢𝘴𝘦 (𝘞5𝘈𝘊𝘔8) gene involved in proline biosynthesis significantly increased in BW4074, which correlated with the iTRAQ data. For BW35695, all six target genes were not differentially expressed, possibly suggesting that the abundances of the proteins and transcripts differentially accumulate at different time points. These qRT-PCR results, call for multi-time point experimental designs for validating iTRAQ data. Overall, the study provides insight into the drought-responsive mechanisms of BW4074 and BW35695 wheat varieties. This information can serve as a reference in studies exploring the differences between plant varieties under drought stress conditions and assist in plant breeding programmes for improved drought resilience.