HURO/0901/1472.2.2 SZEGED - TIMISOARA AXIS FOR THE SAFE FOOD AND FEED

SZETISA1

3. PROGRESS MEETINGTIMISOARA, JANUARY 26-27, 2012

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Horváth Ferenc, Ördög Attila:Effects of Fusarium toxins on K+-channels in

guard cells

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?

Ionic currents through biological membranes

PumpsCarriersIon 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:

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

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 · Po , whereN – number of channels in the cell membranei – current through a single channelPo – open probability

Time-dependent currents are potassium-selective

Two components: Instantenious currents

Time-dependent currents

Whole-cell currents

Patch clamp technique on guard cell protoplasts

Patch clamp configurations

Non-infected maize kernel

extraction

Extraction buffer

Fusarium culmorum

Fusarium graminearum

Maize kernel extracts (by Prof. Erdei)Extraction buffer – acetonitrile: water = 84:16

Experimental solutions

Bath solution:10 mM K-glutamate,1 mM CaCl2,2 mM MgCl2,5 mM MES/KOH (pH 5.5)Osmolarity: 500 mosmol/kg (mannitol)

cell1960 µl

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)

40 µl

50x dilutions from the extracts2% acetonitrile0.4 µg/ml DON

Extraction bufferOutward current: fast reduction after 5 min which remained in the following 20-30’

Inward current: no effect

Water

Outward current: no effect

Inward current: no effect

Negative shift in offset voltage (technical problem)

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

DON (deoxynivalenol)

Outward current: no effect

Inward current: increasing

0.4 µg/ml DON

Extraction from Fusarium graminearum infected maize kernel

Outward current: reduction without restoration

Inward current: reduction without restoration

Extraction from Fusarium culmorum infected maize kernel

Outward current: reduction without restoration

Inward current: reduction without restoration

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