Measurement of lipid peroxidation
Lipid peroxidation as readout for oxidative damage was determined by measuring the reaction product malone dialdehyde (MDA) according to the standard protocols by Heath & Packer (1968) and Hodgson & Raison (1991) with some minor adjustment for grapevine leaves: the leaves (100 mg) were shock-frozen and ground in liquid nitrogen, the powder vortexed for 45 seconds in 1 ml of 0.1 M phosphate buffer (pH 7.4) in a 2.0-ml Eppendorf tube, centrifuged for 4 minutes at 8000 g, and then the sediment discarded. Subsequently, 200 µL of supernatant were added to a reaction mixture containing 750 µL acetic acid (20% w/v), 750 µL 2-thiobarbituric acid (aqueous solution, 0.8% w/v), 200 µL Milli-Q deionised water, and 100 µL sodium dodecyl sulphate (8.1% w/v). An identical reaction mixture, where the supernatant from the sample was replaced by an equal volume of buffer, was used as blank. The reaction mixture was incubated for 1 h at 98 °C, and then cooled down to room temperature. The absorbance at 535 nm (specific signal) and 600 nm (background) were recorded by an ultraviolet spectrophotometer (Uvicon, Schott, Mainz). Lipid peroxidation is then calculated as μM MDA from A535 to A600 using an extinction coefficient of 155 mM-1 cm-1.