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.