Sucrose transporters and sucrose uptake mechanisms in sugarcane

[摘要] ENGLISH ABSTRACT: The process of sugar accumulation and transport in sugarcane is still poorly understood. Understandingthe processes involved in sucrose transport are important, since membrane transport might be importantcontrol points in this pathway. The goals of this project were to unravel the mechanisms of sugartransport in sugarcane culm tissue by using 14C-sugar analysis as well as molecular techniques toidentify 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 discsfrom 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 weremeasured at varying pH, temperature and concentrations of sugars. Hexoses were found to be the majorsugar taken up and sucrose was only important when little hexose was available, as was found in themature ripe internodes. Sucrose uptake differs between tissues and our study showed that sucrose wastaken up rapidly at pH 5, similar to the pH optimum of most sucrose transporters Inhibition studies withTRIS (2-amino-2- (hydroxymethyl)-1,3-propanediol) and PCMBS (p-chloromercuribenzenesulphonicacid) indicated that more than one sucrose transporter activity may be present in the sugarcane systemat 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 strainSEY 6210 (Rae et al., 2004). In an effort to understand sucrose transport in sugarcane culm tissue, apartial sucrose transporter cDNA, ScSUT1(p) from Saccharum hybrid sugarcane a bud cDNA librarywas 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 onnucleotide 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 wasless abundant in immature tissue such as internodes 2 and 3 and in lateral buds. It was concluded thatthe primary function of ScSUT1(p), was not phloem unloading but that the transporter may be involvedin phloem loading, as it is abundant in mature source leaves. ShSUT1 cDNA was obtained from Dr CGrof and the functionality of ShSUT1 as a sucrose transporter in Xenopus leavis oocytes wasconfirmed. However, electrophysiological measurements on the oocytes demonstrated no measurablecurrent associated with sucrose challenge to the oocytes indicating that the transporter activity waseither very low or possibly non-electrogenic. Further investigation is required to characterise thespecific mechanism and kinetic properties of this transporter.

[发布机构] Stellenbosch University