Molecular responses of sorghum cell suspension cultures to high temperature stress
Ngcala, Mamosa Gloria
University of the Free State
High temperatures and frequent drought episodes limit plant growth and development. Ultimately, low crop yields are realised and food insecurity follows. Therefore, there is a need to develop crops that can tolerate extreme temperatures as part of adapting to the global climate change. Sorghum (Sorghum bicolor (L) Moench), a naturally drought tolerant crop, which survives in hot and dry environments was used in this study. The aim of the study was to evaluate the molecular responses of ICSB 338 sorghum cell suspension cultures to high temperature stress. ICSB 338 sorghum cell suspension cultures were exposed to heat stress at 35 and 40oC for 72 hours. Analysis of the cells’ metabolic activity indicated that the cells could survive at both temperatures for 72 hours. However, when Arabidopsis cell suspension cultures were exposed to 40oC for the same period, a significant decrease in cell viability was observed. These results suggest that sorghum is more heat tolerant than Arabidopsis. The proline and glycine betaine content of ICSB 338 cell cultures was determined, following heat stress treatment at 40oC for 72 hours. A decrease in proline content was observed during the stress treatment period. On the other hand, glycine betaine was not detectable at all in the cell culture during the entire stress treatment period. Furthermore, a western blotting experiment was performed to detect the expression pattern of HSP70 in sorghum and Arabidopsis cells in response to heat stress. The results indicated that HSP70 was highly expressed at 40oC in both cell lines. In addition to the metabolic and biochemical changes that occur in plant cells in response to heat stress, protein and gene expression is also altered. Secreted proteins were extracted from the ICSB 338 culture medium, quantified and gel electrophoresed. Furthermore, the differential protein expression analysis of the extracellular matrix (ECM) proteins, following heat stress at 40oC was conducted using isobaric tags for relative and absolute quantitation (iTRAQ) technology. A total of 290 proteins were positively identified. Of these, 231 (80%) were predicted to contain a signal peptide whereas 59 (20%) did not. This indicates that most proteins were targeted for secretion via the classical secretory pathway into the ECM. Of the 290 proteins, 105 were responsive to heat stress with putative functions in metabolism (31%), disease/defence (30%), protein destination and storage (21%), signal transduction (6%) and energy (3%), while 9% had unclear classifications. However, most of the identified proteins (69%) were uncharacterised, possibly indicating their novelty in heat stress response. The expression analysis of ten target heat stress responsive genes from the proteomic dataset and other heat shock marker genes was conducted using quantitative real time-polymerase chain reaction in a time-course experiment (qRT-PCR). For all the genes analysed, differential expression patterns were observed in response to the heat stress. The observed gene and protein expressional changes indicate that sorghum is responsive to heat stress. The knowledge gained could be applied in breeding programmes for the development of heat tolerant crops to alleviate food insecurity in hot and arid regions.
Dissertation (M.Sc. (Botany))--University of the Free State (Qwaqwa Campus), 2018, Molecular responses, High temperatures, Crop development, Climate change, Sorghum