Browsing by Author "Xonti, Alukhanyo"

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    Identification and characterisation of a drought-responsive galactinol synthase in wild rocket (Diplotaxis tenuifolia)
    (Stellenbosch : Stellenbosch University, 2023-12) Xonti, Alukhanyo; Peters, Shaun W.; Loedolff, Bianke; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are sucrosyl-galactosides that occur uniquely in the plant kingdom. The elucidated functional roles attributed to RFOs include serving as carbohydrate transporters, storage reserves and acting as protective agents against biotic and abiotic stress effects. Galactinol synthase (GolS; EC 2.4.1.123) is the key biocatalyst of the RFO biosynthetic pathway and initiates the synthesis of the precursor galactinol (Gol) with the transfer of a galactosyl moiety from UDP-galactose (UDP-Gal) to myo-inositol (Ino). RFO biosynthesis involves the sequential addition of activated galactosyl moieties from Gol to sucrose (Suc) with the enzymes raffinose synthase (RafS; EC 2.4.1.82) and stachyose synthase (StaS; EC 2.4.1.67) to produce raffinose (Raf) and stachyose (Sta) plus higher order RFOs respectively. The coupled transcriptional upregulation of GolS and subsequent RFO accumulation during water deficit stress has been well-studied in several plants; however, these functional dynamics have not been explored in wild rocket (Diplotaxis tenuifolia). The focus of the current study was to isolate and functionally characterise a putative DtGolS1 and investigate its transcriptional regulation and subsequent water-soluble carbohydrates (WSC) profile change in D. tenuifolia leaves under mild water deficit stress. With the heterologous expression of DtGolS1 in E. coli (DH5α), it was demonstrated that the extracts synthesized galactinol in vitro. The predicted DtGolS1 amino acid sequence exhibited GolS hallmarks from other plant species including the C-terminal APSAA pentapeptide and a serine phosphorylating site. Transcriptional analyses also indicated that DtGolS1 is sensitive to water deficit stress as transcript levels observed an upregulation for the most stressed rocket plants. Based on the current findings, the identification of a promising DtGolS1 candidate gene for sugar metabolic manipulation in improving water stress tolerance in wild rocket was revealed. In addition, owing to the prebiotic properties of RFOs, a greater understanding of RFO metabolism particularly in salad greens such as wild rocket could provide insight into biofortification strategies leading to the production of sustainable salad crops.