Wippel K et al. (2010), Inverse pH regulation of plant and fungal sucro...

XB-ART-42084

PLoS One 2010 Aug 05;58:e12429. doi: 10.1371/journal.pone.0012429.

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Inverse pH regulation of plant and fungal sucrose transporters: a mechanism to regulate competition for sucrose at the host/pathogen interface?

Wippel K , Wittek A , Hedrich R , Sauer N .

Abstract
Plant sucrose transporter activities were shown to respond to changes in the extracellular pH and redox status, and oxidizing compounds like glutathione (GSSG) or H(2)O(2) were reported to effect the subcellular targeting of these proteins. We hypothesized that changes in both parameters might be used to modulate the activities of competing sucrose transporters at a plant/pathogen interface. We, therefore, compared the effects of redox-active compounds and of extracellular pH on the sucrose transporters UmSRT1 and ZmSUT1 known to compete for extracellular sucrose in the Ustilago maydis (corn smut)/Zea mays (maize) pathosystem. We present functional analyses of the U. maydis sucrose transporter UmSRT1 and of the plant sucrose transporters ZmSUT1 and StSUT1 in Saccharomyces cerevisiae or in Xenopus laevis oocytes in the presence of different extracellular pH-values and redox systems, and study the possible effects of these treatments on the subcellular targeting. We observed an inverse regulation of host and pathogen sucrose transporters by changes in the apoplastic pH. Under none of the conditions analyzed, we could confirm the reported effects of redox-active compounds. Our data suggest that changes in the extracellular pH but not of the extracellular redox status might be used to oppositely adjust the transport activities of plant and fungal sucrose transporters at the host/pathogen interface.

PubMed ID: 20865151
PMC ID: PMC2928750
Article link: PLoS One

Article Images: [+] show captions

	Figure 1. Effect of different redox reagents on the UmSRT1-mediated sucrose transport in yeast.Uptake was measured in sodium-phosphate buffer pH 5.5 in the presence of the indicated compounds. Cysteine was added to a final concentration of 5 mM, all other compounds to a final concentration of 10 mM (pH-value controlled; nâ=â3Â±SE).
	Figure 2. Effect of different redox reagents on the StSUT1-mediated sucrose transport in yeast.A: Uptake was measured in sodium-phosphate buffer pH 5.5 or pH 7.0 in the presence of the indicated compounds. Cysteine was added to a final concentration of 5 mM, all other compounds to a final concentration of 10 mM. pH 5.5 v â=â vector control. (pH-value controlled; nâ=â3 Â± SD). B: Uptake was measured in sodium-phosphate buffer pH 5.5 in the presence of the indicated compounds that were added from unbuffered solutions. Both unbuffered GSSG (10 mM) and unbuffered GSH (10 mM) reduced the pH-value to 3.3 (nâ=â3Â±SE).
	Figure 3. Effect of the extracellular pH on UmSRT1-mediated sucrose transport in yeast.Uptake was measured in sodium-phosphate buffer pH 5.5 in the presence of the indicated compounds that were added from unbuffered solutions. Both unbuffered GSSG (10 mM) and unbuffered GSH (10 mM) reduced the pH-values of 3.3 (nâ=â3Â±SE).
	Figure 4. Comparison of the pH-dependences of the UmSRT1 sucrose transporter and of the plant sucrose transporters StSUT1 and ZmSUT1.A: Transport rates of UmSRT1 (bars Â± SE) were measured at the indicated pH-values. Measurements from pH 5.0 to pH 8.0 were performed in 50 mM Na+-phosphate buffer, measurements from pH 3.0 to pH 5.0 were performed in 50 mM citrate buffer. The parallel measurements at pH 5.0 in citrate buffer and Na+-phosphate buffer were used to adjust the respective data (nâ€Š=â€Š3). Dotted lines show the pH dependence of the potato sucrose transporter StSUT1 (published in [10]) for comparison. B: pH-dependences of UmSRT1 (light grey bars Â± SE; measured in UmSRT1-expressing yeast cells) and ZmSUT1 (dark grey bars Â± SE; measured in Xenopus oocytes injected with ZmSUT1cRNA; membrane potential: âˆ’100 mV) were determined at the indicated pH values. The pH value yielding the highest transport rate was normalized to 1 (pH 5.0 for UmSRT1; pH 4.5 for ZmSUT1; nâ‰¥3).
	Figure 5. Effect of GSH and GSSG on the StSUT1-mediated sucrose transport in a Îhgt1 deletion mutation.Uptake of 14C-sucrose was measured in sodium-phosphate buffer pH 5.5 in the presence of 10 mM GSG or GSSG. The transport rates in the presence of these compounds are identical to those in Fig. 2 in an Hgt1 wild type strain (pH-values controlled; nâ=â3Â±SE).
	Figure 6. Analysis of the stability of GSH at pH 5.5 at 29Â°C in the presence of yeast cells with Ellman's reagent (DTNB).The amount of GSH was determined with Ellman's reagent after incubation of 10-mM GSH or 10-mM GSSG with yeast cells for 10 min (conditions of the uptake experiments shown in Figures 1, 2 and 4) and compared with the GSH levels measured after mixing the solutions at RT without cells and no further incubation. The data show that the amount of GSH is not significantly reduced during the transport tests (i.e. no GSSG is formed) and also that no GSH is formed from GSSG during the transport analyses (nâ=â3Â±SE).
	Figure 7. Effect of different redox reagents on sucrose-induced currents in ZmSUT1-expressing Xenopus oocytes.A: Currents elicited by 20-mM sucrose in the presence of 0-mM, 1-mM, 5-mM or 10-mM GSSG. B: Currents elicited by 20-mM sucrose in the absence of DTT before [Suc (1)] and after [Suc (2)] a measurement in the presence of 10-mM DTT. C: Currents elicited by 20-mM sucrose in the absence of H2O2 before [Suc (1)] and after [Suc (2)] a measurement in the presence of 0.05% (26.3-mM) H2O2. D: Currents elicited by 20-mM sucrose in the presence of 0-mM, 1-mM, 5-mM or 10-mM GSH. Measurements were performed at pH 5.5 at a holding potential of â70 mV in the presence of the indicated compounds (A: nâ=â6Â±SD; B: nâ=â10Â±SD; C: nâ=â4Â±SD; D: nâ=â3Â±SD).
	Figure 8. Redox-active compounds do not affect plasma membrane targeting of UmSRT1-GFP in baker's yeast.A: Optical section through cells without addition of a redox-active compound. B: Projection of several sections through the same cells as in A. C: Optical section through GSH-treated cells (GSH buffered). D: White-light image of the cells shown in C. E: Projection of several sections through GSSG-treated cells (GSSG unbuffered). F: Projection of several sections through GSH-treated cells (GSH unbuffered). G: Optical section through H2O2-treated cells. H: Projection of several sections through the H2O2-treated cells shown in G. Experiments were performed at an initial pH of 5.5 (25-mM Na+-phosphate buffer). All compounds were added to a final concentration of 10-mM (os â=â optical section; pr â=â projection). Bars are 5 Âµm in all images.

References [+] :

Aoki, Molecular cloning and expression analysis of a gene for a sucrose transporter in maize (Zea mays L.). 1999, Pubmed