HURO/0901/147 2.2.2 Szeged - Timisoara axis for the safe food and feed SZETISA1

1. HURO/0901/1472.2.2Szeged-Timisoara axisfor the safe food and feedSZETISA1 Horváth Ferenc, Ördög Attila:Effects of Fusarium toxins on K+-channels in guard cells 3. Progress Meeting TIMISOARA, January 26-27, 2012 DisclaimerThe content of this page does not necessarily represent the official position of the European Union.

2. Main question Fusarium species produce mycotoxins, predominantly deoxynivalenol (DON), nivalenol, dideoxynivalenol, 3-acetyldeoxynivalenol, zearalenone. DON inhibits the synthesis of DNA and RNA inhibits protein synthesis at the ribosomal level Are there any effect of these toxins on cellular transport processes?

3. Ionic currents through biological membranes Regulated transport processes through biological membranes are prerequisites of life. Pumps Carriers Ion channels (the highest speed = high electric current) Transport through an ion channel is a passive process. Electrochemical potential difference:

4. The voltagedependency of a single-channelcurrent is linear Ohm’s law: I = V/R Slope = 1/R = g (conductance) [pS] Current direction: negative current – cation influx or anion efflux positive current – cation efflux or anion influx

5. Gatingfunction of channels Two conformation states: open and closed. The distribution between the two states depends on physical and chemical factors. Whole cell current: I = N · i · Po, where N – number of channels in the cell membrane i – current through a single channel Po – open probability

6. Whole-cell currents Two components: Instantenious currents Time-dependent currents Time-dependent currents are potassium-selective

7. Patch clamp technique on guard cell protoplasts Patch clamp configurations

8. Maize kernel extracts (by Prof. Erdei) Extraction buffer – acetonitrile: water = 84:16 Extractionbuffer Non-infectedmaize kernel extraction Fusarium graminearum Fusarium culmorum

9. Experimental solutions Pipette solution (cytosol): 150 mM K-glutamate, 0.838 mM CaCl2, 2 mM MgCl2, 2 mM K-ATP, 1 mM EGTA, 10 mM HEPES/KOH (pH 7.5) Cytosolic free Ca2+ concentration 200 nM (calculation according to Föhr et al. 1992). Osmolarity: 520 mosmol/kg (mannitol) 50x dilutions from the extracts 2% acetonitrile 0.4 µg/ml DON cell 1960 µl 40 µl Bath solution: 10 mM K-glutamate, 1 mM CaCl2, 2 mM MgCl2, 5 mM MES/KOH (pH 5.5) Osmolarity: 500 mosmol/kg (mannitol)

10. Outward current: fast reduction after 5 min which remained in the following 20-30’ Inward current: no effect Extraction buffer

11. Water Outward current: no effect Inward current: no effect Negative shift in offset voltage (technical problem)

12. Extraction from non-infected maize kernel Outward current: fast reduction after 5 min which remained in the following 20-30’ Inward current: fast increase that returned after 30 min

13. DON (deoxynivalenol) 0.4 µg/ml DON Outward current: no effect Inward current: increasing

14. Extraction from Fusarium graminearum infected maize kernel Outward current: reduction without restoration Inward current: reduction without restoration

15. Extraction from Fusarium culmorum infected maize kernel Outward current: reduction without restoration Inward current: reduction without restoration

16. Summary Extraction buffer 0.4 µg/ml DON Extraction from non-infected maize kernel Extraction from FG- or FC-infected maize kernel Outward current: fast reduction after 5 min which remained in the following 20-30’ Inward current: no effect Outward current: no effect Inward current: increasing Outward current: fast reduction after 5 min which remained in the following 20-30’ Inward current: fast increase that returned after 30 min Outward current: reduction without restoration Inward current: reduction without restoration DON restores the decreasing effect of acetonitrile on the outward current DON also increases the inward current Maize kernel extract activates the inward current, that returns after 30 min FG and FC-infected kernel extracts inhibit the inward current