Characterisation of the physiological, biochemical and molecular responses of sorghum to drought stress
University of the Free State
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.
Thesis (Ph.D. (Botany))--University of the Free State (Qwaqwa Campus), 2018, Sorghum, Arabidopsis, Drought stress, Proteomics, Cell suspension culture, 2D-DiGE, iTRAQ, qRT-PCR, Gene expression, Drought markers