Sucrose transporters and sucrose uptake mechanisms in sugarcane

Titus, Charlene H. A. (Charlene Helecyn Agatha) (Stellenbosch : University of Stellenbosch, 2004-12)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: The process of sugar accumulation and transport in sugarcane is still poorly understood. Understanding the processes involved in sucrose transport are important, since membrane transport might be important control points in this pathway. The goals of this project were to unravel the mechanisms of sugar transport in sugarcane culm tissue by using 14C-sugar analysis as well as molecular techniques to identify possible sucrose transporters. Developing (internode 2 and 4) and maturing (internode 8 and 15) culm tissue of sugarcane (Saccharum hybrid) commercial variety N19 was used for all tissue disc experiments. Tissue discs from internodes of different developmental stages were cut from field grown sugarcane plants (cv. N19) and the uptake of 14C-labelled glucose, fructose and sucrose measured. The uptake rates were measured at varying pH, temperature and concentrations of sugars. Hexoses were found to be the major sugar taken up and sucrose was only important when little hexose was available, as was found in the mature ripe internodes. Sucrose uptake differs between tissues and our study showed that sucrose was taken up rapidly at pH 5, similar to the pH optimum of most sucrose transporters Inhibition studies with TRIS (2-amino-2- (hydroxymethyl)-1,3-propanediol) and PCMBS (p-chloromercuribenzenesulphonic acid) indicated that more than one sucrose transporter activity may be present in the sugarcane system at different sucrose concentrations. To date work on sugarcane sucrose transporter expression on DNA and RNA level has been limited. Only recently a sucrose transporter from Saccharum hybrid sugarcane stem cDNA libray, ShSUT1 (Saccharum hybrid Sucrose Transporter ) was isolated and functionally characterized in the yeast strain SEY 6210 (Rae et al., 2004). In an effort to understand sucrose transport in sugarcane culm tissue, a partial sucrose transporter cDNA, ScSUT1(p) from Saccharum hybrid sugarcane a bud cDNA library was isolated, and cloned from a bud cDNA library. The clone was designated ScSUT(p) as a partial Sugarcane Sucrose Transporter. The ScSUT1(p) sequence showed 94% identity to ShSUT1 on nucleotide level over 1258 nucleotides and had an estimated open reading frame of 419 amino acids. Southern blot analysis indicated that the transporter had a low copy number and the ScSUT1(p) transcript expression was constitutive in sucrose accumulating and sucrose storing stem tissue, but was less abundant in immature tissue such as internodes 2 and 3 and in lateral buds. It was concluded that the primary function of ScSUT1(p), was not phloem unloading but that the transporter may be involved in phloem loading, as it is abundant in mature source leaves. ShSUT1 cDNA was obtained from Dr C Grof and the functionality of ShSUT1 as a sucrose transporter in Xenopus leavis oocytes was confirmed. However, electrophysiological measurements on the oocytes demonstrated no measurable current associated with sucrose challenge to the oocytes indicating that the transporter activity was either very low or possibly non-electrogenic. Further investigation is required to characterise the specific mechanism and kinetic properties of this transporter.

AFRIKAANSE OPSOMMING: Die proses van suikerakkumulering en -vervoer in suikerriet word steeds baie vaag verstaan. ‘n Deeglike begrip van die prosessewat betrokke is in die vervoer van sukrose is baie belangrik omdat transmembraan vervoer moontlik een van die belangrike beheerpunte in metabolisme mag wees. Die doelwitte van die studie was om ‘n beter begrip te bekom van die meganisme wat betrokke is by die vervoer en berging van sukrose in suikerriet. Die projek is in ‘n fisiologiese en ‘n molekulêre afdeling verdeel. In die fisiologiese afdeling is stingelweefsel van ‘n Saccharum hybried (variëteit N19) van verskillende stadiums van ontwikkeling (internodes 2-4, internode 8 en internode 15) gebruik. Opname van radioaktiewe (14C) sukrose, glukose en fruktose is as analise metode gebruik vir die suikeropname eksperimente. Die invloed van pH, suiker konsentrasie en inhibitore soos PCMBS (pchloromercuriphenylsulfonic acid) en TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) op die tempo van suikeropname is ondersoek. Die molekulêre deel fokus hoofsaaklik op die identifisering, isolering en karakterisering van nuwe sukrose vervoerproteine in suikerriet, met behulp van PCR en heteroloë uitdrukking in Xenopus laevis oösiete. Die 14C - opname eksperimente het tot die volgende gevolgtrekkings gelei: Heksoses speel die belangrikste rol in die vervoer van suiker in die riet as daar min of geen sukrose teenwoordig is nie. Sodra daar sukrose in groot mate teenwoordig is soos in die geval van ontwikkelde, ryp internodes, is die rol van sukrose egter belangriker. Sukrose is die maklikste opgeneem by pH 5, wat naby die pH optimum van die meeste sukrose vervoerproteïene is. TRIS en PCMBS het beide ‘n inhiberende effek op sukrose opname gehad, maar die invloed was groter by die laer sukrose konsentrasies. Tot onlangs was daar baie min inligting oor sukrose vervoer in suikerriet op DNA en RNA vlak. Die eerste sukrose vervoerprotein uit suikerriet, ShSUT1 (Saccharum Hibried Sukrose Transporter) is eers onlangs uit ‘n stingel - cDNA biblioteek geïsoleer (Rae et al., 2004) en die funksionering daarvan is in ‘n gisras (SEY6210) getoets. In my pogings om sukrose vervoer te verstaan is ‘n gedeeltelike cDNA, naamlik ScSUT(p) (partial Sugarcane Sucrose Transporter) van 1258 nukleotiede, uit cDNA afkomstig van suikerrietbotsel geïsoleer. Die nukleotiedvolgorde stem 94% ooreen met ShSUT1 en kodeer vir ‘n moontlike oopleesraam van 419 aminosure. Southern analises het aangedui dat ScSUT(p) ‘n lae kopie getal het, in ooreenstemming met wat vir ander sukrose vervoerproteïene gevind is. Northern analises het getoon dat die uitdrukking van ScSUT(p) konstitutatief is in sukrose akkumulerende sowel as sukrose bergingsweefsel. Jong weefsel (internode 2 en 3) het baie lae uitdrukking getoon, met die hoogste uitdrukking in blaarweefsel. Uit die resultate is afgelei dat ScSUT(p) ‘n rol in floeëmlading en -ontlading mag speel. Xenopus laevis oösiete, is as ‘n heteroloë uitdrukking sisteem gebruik om te bevestig dat ShSUT1 as ‘n sukrose vervoerproteïen funksioneer. Elektrofisiologie het nie daarin geslaag om ShSUT1 se spesifieke werkingsmeganisme te identifiseer nie. Aanduidings is egter gevind dat ShSUT1 moontlik nie as ‘n H+/sukrose simportsisteem werk nie, maar by gefasilliteerde vervoer van sukrose betrokke mag wees. Verdere navorsing is noodsaaklik om die meganisme van ShSUT1 se werking te verstaan.

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