1. HURO/0901/1472.2.2 SZEGED - TIMISOARA AXIS FOR THE SAFE FOOD AND FEED SZETISA1 3. PROGRESS MEETING TIMISOARA, JANUARY 26-27, 2012 Disclaimer The content of this page does not necessarily represent the official position of the European Union. Horváth Ferenc, Ördög Attila: Effects of Fusarium toxins on K + -channels in guard cells

2. 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 Main question Are there any effect of these toxins on cellular transport processes?

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

4. The voltage dependency of a single-channel current 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. Gating function 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 · P o, where N – number of channels in the cell membrane i – current through a single channel P o – open probability

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

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

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

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

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

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) Outward current: no effect Inward current: increasing 0.4 µg/ml DON

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 Outward current: fast reduction after 5 min which remained in the following 20-30’ Inward current: no effect 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 Outward current: reduction without restoration Inward current: reduction without restoration Extraction from FG- or FC- infected maize kernel Outward current: no effect Inward current: increasing 0.4 µg/ml DON 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