Browsing by Author "Hugo, Melt"

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    Functional identification of a putative stachyose synthase (StaS, Medtr7g106910.1) from Medicago truncatula, by overexpression in the Arabidopsis stachyose deficient double mutant atrs4/atrs5
    (Stellenbosch : Stellenbosch University, 2018-11) Hugo, Melt; Peters, Shaun W.; Guzha, Tapiwa; Loedolff, Bianke; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: The Raffinose Family Oligosaccharides (RFOs; Suc-Galn, 13 < n ≥ 1) are α1,6- galactosyl extensions of sucrose occurring exclusively in the plant kingdom and some photoautotrophic bacteria. This unique group of sugars are widely implicated as storage compounds in sink tissues, phloem translocates, and as target molecules that help combat abiotic and biotic plant stresses in various species. The RFO biosynthetic pathway is well characterised and RFOs are synthesised from sucrose by the successive addition of galactose moieties by α-1,6 galactosyltransferases viz. galactinol synthase (GolS, EC 2.4.1.123), raffinose synthase (RafS, EC 2.4.1.82), and stachyose synthase (StaS, EC 2.4.1.67). Amino acid sequence alignments between functionally identified RafS and StaS proteins indicate that the major difference between them is the presence of a conserved motif between amino acid positions 340 to 420 (absent in RafS proteins). The predicted protein sequence of StaS from the model legume - Medicago trancatula, Medtr7g106910.1 (designated MtStaS) contains this motif. To explore the functional identity of these carbohydrates in legumes, cDNA encoding stachyose synthase (StaS) which transfers a galactosyl moiety from galactinol to the C6 position of the galactose moiety in raffinose (Raf) to yield the tetrasaccharide stachyose (Sta), was identified and cloned from M. truncatula. As part of a multipronged strategy to functionally characterise MtStaS, we performed the following experiments. We (i) identified a candidate MtStaS gene through rudimentary bioinformatic analyses. We then examined MtStaS transcript abundance in a variety of M. truncatula organs and concluded that MtStaS expression is tissue-specific ii) cloned the candidate gene into a binary vector pMDC32 (dual CaMV35s promoter) and transformed this construct into the Arabidopsis thaliana atrs4 (devoid of detectable Sta) and atrs4.atrs5 (devoid of detectable Raf and Sta) TDNA insertion mutants in an attempt to restore the RFO metabolism. We confirmed that MtStaS is able to recover ablated Sta in atrs4 mature seeds and (iii) cloned MtStaS and subsequently characterised it in the dimorphic fungus - Yarowia lipolytica. We established that it is a bona fide StaS that possess no bifunctionality in synthesising both Raf and Sta, contradictory to StaS from Arabidopsis thaliana (AtStaS) which can synthesise both.