Browsing Masters Degrees (Genetics) by Author "Barker, Adele Mariska"
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- ItemThe over-expression of a late embryogenesis abundant protein in sugarcane to improve water-deficit stress tolerance.(Stellenbosch : Stellenbosch University, 2021-03) Barker, Adele Mariska; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Understanding the underlying mechanisms behind the acquisition of desiccation tolerance in the vegetative tissues of resurrection plants may serve as a blueprint for generating crops with enhanced drought tolerance. Analysis of water-deficit stress responses in Xerophyta humilis, a small resurrection plant endemic to South Africa, found that the late embryogenesis abundant (LEA) protein called XhLEA accumulated in the desiccated leaf tissues of the plant. LEA proteins have been shown to play a protective role against damage caused by environmental stresses and may play a critical role in the desiccation- tolerant phenotype of X. humilis. Here, we characterized the novel group 1 LEA gene named XhLEA from X. humilis. It has been shown to be highly expressed in the dehydrated leaf tissues of the plant. XhLEA shares the highest nucleotide sequence homology with group 1 LEA genes found in monocotyledonous plants that are members of the Poaceae family. This study aimed to overexpress XhLEA in transgenic sugarcane (Saccharum spp. hybrid cv. NCo310) in an attempt to enhance drought tolerance in the transgenic plants. XhLEA was first cloned independently into a constitutively-expressed (pUbi510:XhLEA) and a drought-inducible (pAHC20:Rab17:XhLEA) plant expression vector, and subsequently transformed in sugarcane using a biolistic particle approach. Transgenic plants were regenerated in vitro and selected using geneticin. The plants surviving the selection phase were hardened off in the greenhouse and putative transgenic lines were analysed for transgene insertion and expression. One and three-month-old wild type (WT) and transgenic plants were exposed to drought in glasshouse pot trials. Transgenic plants had a lower rate of water loss under water-deficit conditions as indicated by the relative water content (RWC) and had a higher level of water retention under non-stressed conditions when compared to the WT. Physiological measurements indicated that higher levels of stomatal conductance and photosystem II efficiency were maintained for longer in the transgenic plants exposed to stress. Proteins related to the photosynthetic activity of the plants, such as pyruvate orthophosphate dikinase (PPDK), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), and phosphoenolpyruvate carboxylase (PEPC) were also significantly upregulated in transgenic plants under water-deficit conditions. Biochemical analysis of the 3-month-old plants exposed to drought indicated that the reactive oxygen species (ROS) such as H2O2 and O⁻, and malondialdehyde (MDA) accumulation were similar among all genotypes. This was in contrast with the significantly lower levels of H2O2 accumulation that was observed in the 1-month-old stressed transgenic sugarcane plants compared to the WT plants. Furthermore, membrane electrolyte leakage (EL) was significantly lower in the stressed 3-month-old transgenic lines when compared to the WT plants but no marked difference was observed in reduced glutathione (GSH) content or in the activities of ROS scavenging enzymes such as superoxide dismutase (SOD) and catalase (CAT) across all the lines. Accumulation of osmolytes such as proline was affected by the XhLEA overexpression, however, the responses varied with the T1 and T2 lines being either significantly higher or lower than the WT sugarcane, respectively. Sub-cellular localization studies, using an YFP-XhLEA fusion protein, indicated that protein expression in the epidermis of red onion cells was likely localized in the nucleus and cell membrane. Overall, overexpression of XhLEA enhances drought tolerance mainly through the maintenance of vital cellular processes, such as photosynthesis. However, constitutive expression of the XhLEA transgene also resulted in growth impairment as well as reduced sucrose accumulation in mature sugarcane grown under non-stressed conditions. Future analysis of the drought-inducible XhLEA-expressing plants is required to determine if the reduced growth yield phenotype observed in the transgenic plants overexpressing XhLEA in a constitutive manner can be avoided.