Advertisement

Phloem pp 253-266 | Cite as

Measuring Sucrose Transporter Activities Using a Protoplast-Esculin Assay

  • Theresa Rottmann
  • Ruth StadlerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2014)

Abstract

Sucrose transport across membranes requires the activity of transport proteins. Sucrose-specific SWEET proteins mediate sugar efflux out of the cytosol and SUC proteins catalyze the uptake of sucrose from the apoplast. Both transport processes are involved in phloem loading in source leaves as well as in the post-phloem pathway in sink tissues. An important step during the characterization of new sucrose transporters is to analyze their transport activity. This is usually achieved by heterologous expression of the respective gene in yeast cells or Xenopus oocytes and subsequent uptake measurements. However, in some cases, mistargeting to internal membranes or the lack of protein modifications and/or interaction partners in the heterologous system can interfere with uptake analyses. Therefore, a new in planta method was developed that is based on mesophyll protoplasts as expression system and the fluorescent sucrose analog esculin to monitor uptake activities by confocal microscopy. In this chapter we describe the design of constructs required to analyze sucrose transporters in protoplasts, the experimental setup of the protoplast-esculin assay, and the quantitative evaluation of the obtained data. The quantification of esculin uptake allows the application of the new assay to a variety of questions, e.g., by comparison of point mutants, splice variants, or transporters with and without interaction partners.

Key words

Sucrose Sugar transport proteins Uptake measurement Esculin Protoplasts Confocal microscopy Phloem 

References

  1. 1.
    Haritatos E, Medville R, Turgeon R (2000) Minor vein structure and sugar transport in Arabidopsis thaliana. Planta 211:105–111CrossRefGoogle Scholar
  2. 2.
    Sauer N (2007) Molecular physiology of higher plant sucrose transporters. FEBS Lett 581:2309–2317CrossRefGoogle Scholar
  3. 3.
    Gamalei Y (1989) Structure and function of leaf minor veins in trees and herbs. Trees 3:96–110CrossRefGoogle Scholar
  4. 4.
    Chen L-Q, Qu X-Q, Hou B-H et al (2012) Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science 335:207–211CrossRefGoogle Scholar
  5. 5.
    Marger MD, Saier MH (1993) A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci 18:13–20CrossRefGoogle Scholar
  6. 6.
    Zhou J-J, Theodoulou F, Sauer N et al (1997) A kinetic model with ordered cytoplasmic dissociation for SUC1, an Arabidopsis H+ /sucrose cotransporter expressed in Xenopus oocytes. J Membr Biol 159:113–125CrossRefGoogle Scholar
  7. 7.
    Lemoine R (2000) Sucrose transporters in plants: update on function and structure. Biochim Biophys Acta Biomembr 1465:246–262CrossRefGoogle Scholar
  8. 8.
    Williams LE, Lemoine R, Sauer N (2000) Sugar transporters in higher plants – a diversity of roles and complex regulation. Trends Plant Sci 5:283–290CrossRefGoogle Scholar
  9. 9.
    Sauer N, Ludwig A, Knoblauch A et al (2004) AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes. Plant J 40:120–130CrossRefGoogle Scholar
  10. 10.
    Chen L, Hou B, Lalonde S et al (2010) Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468:527–532CrossRefGoogle Scholar
  11. 11.
    Stadler R, Sauer N (1996) The Arabidopsis thaliana AtSUC2 gene is specifically expressed in companion cells. Bot Acta 109:299–306CrossRefGoogle Scholar
  12. 12.
    Schneider S, Schneidereit A, Udvardi P et al (2007) Arabidopsis INOSITOL TRANSPORTER2 mediates H+ symport of different inositol epimers and derivatives across the plasma membrane. Plant Physiol 145:1395–1407CrossRefGoogle Scholar
  13. 13.
    Rottmann TM, Klebl F, Schneider S et al (2018) Sugar transporter STP7 specificity for L-arabinose and D-xylose contrasts with the typical hexose transporters STP8 and STP12. Plant Physiol 176:2330–2350CrossRefGoogle Scholar
  14. 14.
    Weise A, Barker L, Kühn C et al (2000) A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants. Plant Cell 12:1345–1355CrossRefGoogle Scholar
  15. 15.
    Schneider S, Hulpke S, Schulz A et al (2012) Vacuoles release sucrose via tonoplast-localised SUC4-type transporters. Plant Biol 14:325–336CrossRefGoogle Scholar
  16. 16.
    Rottmann TM, Fritz C, Lauter A et al (2018) Protoplast-esculin assay as a new method to assay plant sucrose transporters: Characterization of AtSUC6 and AtSUC7 sucrose uptake activity in Arabidopsis Col-0 ecotype. Front Plant Sci 9:430.  https://doi.org/10.3389/fpls.2018.00430CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gora PJ, Reinders A, Ward JM (2012) A novel fluorescent assay for sucrose transporters. Plant Methods 8:13.  https://doi.org/10.1186/1746-4811-8-13CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Nieberl P, Ehrl C, Pommerrenig B et al (2017) Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves. Plant Biol 19:315–326CrossRefGoogle Scholar
  19. 19.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefGoogle Scholar
  20. 20.
    Dotzauer D, Wolfenstetter S, Eibert D et al (2010) Novel PSI domains in plant and animal H+-inositol symporters. Traffic 11:767–781CrossRefGoogle Scholar
  21. 21.
    Curtis MD, Grossniklaus U (2003) A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Molecular Plant Physiology, Department of BiologyFriedrich-Alexander University Erlangen-NurembergErlangenGermany

Personalised recommendations