Functional identification of a putative stachyose synthase (StaS, Medtr7g106910.1) from Medicago truncatula, by overexpression in the Arabidopsis stachyose deficient double mutant atrs4/atrs5

Hugo, Melt (2018-11)

Thesis (MScAgric)--Stellenbosch University, 2018.

Thesis

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.

AFRIKAANSE OPSOMMING: Raffinose Familie Oligosakkariede (RFOs; Suc-Galn, 13 < n ≥ 1) is α1,6-galaktosiel verlengings van sukrose wat slegs in die plant koninkryk en ‘n seleksie van fotooutotrofiese bakterieë voorkom. Die unieke suikers vervul kritiese rolle in plante en dien as storing molekule in die sink weefsel, floëem translokasie molekules, en as teiken molekule wat abiotiese en biotiese spanning teen te veg. Die RFO biosintetiese padweg is goed gekarakteriseer en RFOs word geproduseer vanaf sucrose deur die opeenvolgende byvoeging van galaktosiel molekules deur α-1,6 galaktosielltransferases viz. galaktinol sintase (GolS, EC 2.4.1.123), raffinose sintase (RafS, EC 2.4.1.82), en stachyose sintase (StaS, EC 2.4.1.67). Vergelykings tussen die aminosuur volgordes van funksionele RafS en StaS proteïne toon groot ooreenstemming behalwe vir ‘n bewaarde gebied tussen posisie 330 en 410 wat afwesig is van RafS proteïne. Die gebied is teenwoordig in die vermeende proteïen volgorde van die StaS van Medicago trancatula, Medtr7g106910.1 (aangewese MtStaS). Stachyose sintase (StaS) word vermoed om tetrasakariede stachyose (Sta) te produseer deur die oordrag van ‘n galaktosiel molekule vanaf galaktinol na raffinose (Raf) te kataliseer. Om die funksionele rol van die suikers in peulplante verder te ondersoek was stachyose sintase (StaS) vanaf die kDNS van M. truncatula geïdentifiseer en gekloneer. Om MtStaS ten volle funksioneel te karakteriseer was die volgende eksperimente uitgevoer. Ons het (i) die kandidaat MtStaS geen deur rudimentêre bioinformatiese analise geïdentifiseer. Die hoeveelheid MtStaS geen uitdrukking in verskeie M. truncatula organe was ook bepaal, wat aangedui het MtStaS geen uitdrukking is weefsel spesifiek. Daarna is (ii) MtStaS gekloneer in pMDC32 (dubbele CaMV35s promotor) en die konstruk getransformeer in Arabidopsis thaliana atrs4 (geen waarneembare Sta) en atrs4.atrs5 (geen waarneembare Raf en Sta) om sodoende te bepaal of RFO metabolisme herstel kon word. Ons het bevestig dat atrs4 mutante wat gekomplimenteer is met MtStaS wel Sta in sade kan produseer. Laastens was (iii) MtStaS ook in uitgedruk en gekarakteriseer in Yarowia lipolytica. Ons het bevestig dat MtStaS ‘n bona fide StaS is wat slegs Sta produseer en nie bifunksionaliteit toon soos die StaS van Arabidopsis thaliana (AtStaS).

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