Plant Molecular Biology

, Volume 44, Issue 5, pp 687–697 | Cite as

Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization

  • Michael L. Gear
  • Mary L. McPhillips
  • John W. Patrick
  • David W. McCurdy

Abstract

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H+/hexose symporter. The Km of the symporter for glucose is 45 μM.

gene expression heterologous expression H+/hexose symporter Lycopersicon esculentum quantitative PCR Saccharomyces cerevisiae 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Asano, T., Katagiri, H., Takata, K., Lin, J.L., Ishihara, H., Inukai, K., Tsukuda, K., Kikuchi, M., Hirano, H., Yazaki, Y. and Oka, Y. 1991. The role of N-glycosylation of GLUT1 for glucose transport activity. J. Biol. Chem. 266: 24632–24636.PubMedGoogle Scholar
  2. Boorer, K.J., Loo, D.D.F. and Wright, E.M. 1994. Steady-state and presteady-state kinetics of the HC/hexose cotransporter (STP1) from Arabidopsis thaliana expressed in Xenopus oocytes. J. Biol. Chem. 269: 20417–20424.PubMedGoogle Scholar
  3. Bugos, R.C. and Thom, M. 1993. Glucose transporter cDNAs from sugarcane. Plant Physiol. 103: 1469–1470. Bush, D.R. 1993. Proton-coupled sugar and amino acid transporters in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 513– 542.Google Scholar
  4. Büttner, M., Truernit, E., Baier, K., Scholz-Starke, J., Sontheim, M., Lauterbach, C., Huss, V.R. and Sauer, N. 2000. AtSTP3, a green leaf-specific, low affinity monosaccharide-HC symporter of Arabidopsis thaliana. Plant Cell Environ. 23: 175–184.Google Scholar
  5. Caspari, T., Robl, I., Stolz, J. and Tanner, W. 1996. Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro. Plant J. 10: 1045–1053.PubMedGoogle Scholar
  6. Elble, R. 1992. A simple and efficient procedure for transformation of yeasts. Biotechniques 13: 18–20.PubMedGoogle Scholar
  7. Felle, H., Gogarten, J.P. and Bentrup, F.W. 1983. Phlorizin inhibits hexose transport across the plasmalemma of Riccia fluitans. Planta 157: 267–270.Google Scholar
  8. Getz, H.P., Knauer, D. and Willenbrink, J. 1987. Transport of sugar across the plasma membrane of beetroot protoplasts. Planta 171: 185–196.Google Scholar
  9. Gogarten, J.P. and Bentrup, F.W. 1989. Substrate specificity of the hexose carrier in the plasmalemma of Chenopodium rubrum suspension cells probed by transmembrane exchange diffusion. Planta 178: 52–60.Google Scholar
  10. Harrison, M.J. 1996. A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations. Plant J. 9: 491–503.PubMedGoogle Scholar
  11. Kephart, D. 1998. Quantitative RT-PCR: rapid construction of templates for competitive amplification. Promega Notes 68: 16–19.Google Scholar
  12. Köhler, T. 1995. General aspects and chances of nucleic acid quantitation by PCR. In: T. Köhler, D. Lassner, A.-K. Rost, B. Thamm, B. Pustowoit and H. Remke (Eds) Quantitation of mRNA by Polymerase Chain Reaction, Springer-Verlag, Berlin, pp. 3–14.Google Scholar
  13. Lalonde, S., Boles, E., Hellmann, H., Barker, L., Patrick, J.W., Frommer, W.B. and Ward, J.M. 1999. The dual function of sugar carriers: transport and sugar sensing. Plant Cell 11: 707–726.PubMedGoogle Scholar
  14. Lashbrook, C.C., Gonzalez-Bosch, C. and Bennett, A.B. 1994. Two divergent endo-β-1,4-glucanase genes exhibit overlapping expression in ripening fruit and abscising flowers. Plant Cell 6: 1485–1493.CrossRefPubMedGoogle Scholar
  15. Lemoine, R. and Delrot, S. 1987. Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves. Physiol. Plant. 69: 639–644.Google Scholar
  16. Lin, W., Schmitt, M.R., Hitz, W.D. and Giaquinta, R.T. 1984. Sugar transport in isolated corn root protoplasts. Plant Physiol. 76: 894–897.Google Scholar
  17. Loison, G. 1994. Production of foreign proteins at high level. In: J.R. Johnston (Ed.) Molecular Genetics of Yeast: A Practical Approach, Oxford University Press, Oxford, pp. 161–180.Google Scholar
  18. Maiden, M.C.J., Davis, E.O., Baldwin, S.A., Moore, D.C.M. and Henderson, P.J.F. 1987. Mammalian and bacterial sugar transport proteins are homologous. Nature 325: 641–643.PubMedGoogle Scholar
  19. Marger, M.D. and Saier, M.H. 1993. A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem. Sci. 18: 13–20.PubMedGoogle Scholar
  20. Martinoia, E., Kaiser, G., Schramm, M.J. and Heber, U. 1987. Sugar transport across the plasmalemma and the tonoplast of barley mesophyll protoplasts. Evidence for different transport systems. J. Plant Physiol. 131: 467–478.Google Scholar
  21. Maynard, J.W. and Lucas, W.J. 1982. Sucrose and glucose uptake into Beta vulgaris leaf tissues. A case for general (apoplastic) retrieval systems. Plant Physiol. 70: 1436–1443.Google Scholar
  22. Opekarova, M., Caspari, T. and Tanner, W. 1994. The HUP1 gene product of Chlorella kessleri: HC/glucose symport studied in vitro. Biochim. Biophys. Acta 1194: 149–154.PubMedGoogle Scholar
  23. Reifenberger, E., Freidel, K. and Ciriacy, M. 1995. Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol. Microbiol. 16: 157–167.PubMedGoogle Scholar
  24. Rentsch, D., Boorer, K.J. and Frommer, W.B. 1998. Structure and function of plasma membrane amino acid, oligopeptide and sucrose transporters from higher plants. J. Membr. Biol. 162: 177–190.PubMedGoogle Scholar
  25. Roitsch, T. and Tanner, W. 1994. Expression of a sugartransporter gene family in a photoautotrophic suspension culture of Chenopodium rubrum L. Planta 193: 365–371.CrossRefPubMedGoogle Scholar
  26. Ruan, Y.L. and Patrick, J.W. 1995. The cellular pathway of postphloem sugar transport in developing tomato fruit. Planta 196: 434–444.Google Scholar
  27. Ruan, Y.L., Patrick, J.W. and Brady, C. 1997. Protoplast hexose carrier activity is a determinate of genotypic difference in hexose storage in tomato fruit. Plant Cell Environ. 20: 341–349.Google Scholar
  28. Sauer, N. and Stadler, R. 1993. A sink-specific HC/monosaccharide co-transporter from Nicotiana tabacum: cloning and heterologous expression in baker's yeast. Plant J. 4: 601–610.PubMedGoogle Scholar
  29. Sauer, N., Caspari, T., Klebl, F. and Tanner, W. 1990a. Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA 87: 7949–7952.PubMedGoogle Scholar
  30. Sauer, N., Friedlander, K. and Graml Wicke, U. 1990b. Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana. EMBO J. 9: 3045–3050.PubMedGoogle Scholar
  31. Sauer, N., Baier, K., Gahrtz, M., Stadler, R., Stolz, J. and Truernit, E. 1994. Sugar transport across the plasma membranes of higher plants. Plant Mol. Biol. 26: 1671–1679.PubMedGoogle Scholar
  32. Smith, F.W., Ealing, P.M., Hawkesford, M.J. and Clarkson, D.T. 1995. Plant members of a family of sulfate transporters reveal functional subtypes. Proc. Natl. Acad. Sci. USA 92: 9373–9377.Google Scholar
  33. Stadler, R., Wolf, K., Hilgarth, C., Tanner, W. and Sauer, N. 1995. Subcellular localization of the inducible Chlorella HUP1 monosaccharide-HC symporter and cloning of a co-induced galactose-HC symporter. Plant Physiol. 107: 33–41.PubMedGoogle Scholar
  34. Stanzel, M., Sjölund, R.D. and Komor, E. 1988. Transport of glucose, fructose and sucrose by Streptanthus tortuosus suspension cells. I. Uptake at low sugar concentration. Planta 174: 201–209.Google Scholar
  35. Tanner, W. and Caspari, T. 1996. Membrane transport carriers. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 595–626.PubMedGoogle Scholar
  36. Truernit, E., Schmid, J., Epple, P., Illig, J. and Sauer, N. 1996. The sink-specific and stress-regulated Arabidopsis STP4 gene: enhanced expression of a gene encoding a monosaccharide transporter by wounding, elicitors, and pathogen challenge. Plant Cell 8: 2169–2182.PubMedGoogle Scholar
  37. Truernit, E., Stadler, R., Baier, K. and Sauer, N. 1999. A male gametophyte-specific monosaccharide transporter in Arabidopsis. Plant J. 17: 191–201.PubMedGoogle Scholar
  38. Tubbe, A. and Buckhout, T.J. 1992. In vitro analysis of the HChexose symporter on the plasma membrane of sugarbeets (Beta vulgaris L.). Plant Physiol. 99: 945–951.Google Scholar
  39. Verstappen, R., Ranostaj, S. and Rausch, T. 1991. The hexose transporters at the plasma membrane and the tonoplast of transformed plant cells: kinetic characterization of two distinct carriers. Biochim. Biophys. Acta 1073: 366–373.PubMedGoogle Scholar
  40. Weber, H., Borisjuk, L., Heim, U., Sauer, N. and Wobus, U. 1997. A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds. Plant Cell 9: 895–908.PubMedGoogle Scholar
  41. Weig, A., Franz, J., Sauer, N. and Komor, E. 1994. Isolation of a family of cDNA clones from Ricinus communis L. with close homology to the hexose carriers. J. Plant Physiol. 143: 178–183.Google Scholar
  42. Will, A., Caspari, T. and Tanner, W. 1994. Km mutants of the Chlorella monosaccharide/HC cotransporter randomly generated by PCR. Proc. Natl. Acad. Sci. USA 91: 10163–10167.PubMedGoogle Scholar
  43. Xia, J.H. and Saglio, P.H. 1988. Characterization of the hexose transport system in maize root tips. Plant Physiol. 88: 1015– 1020.Google Scholar
  44. Ylstra, B., Garrido, D., Busscher, J. and van Tunen, A.J. 1998. Hexose transport in growing petunia pollen tubes and characterization of a pollen-specific, putative monosaccharide transporter. Plant Physiol. 118: 297–304.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Michael L. Gear
    • 1
  • Mary L. McPhillips
    • 1
  • John W. Patrick
    • 1
  • David W. McCurdy
    • 1
  1. 1.Department of Biological SciencesThe University of NewcastleNewcastleAustralia

Personalised recommendations