2.4.2 Lipid peroxidation
We evaluated lipid peroxidation of the plasma membrane as a proxy of the level of oxidative damage in plant tissues grown under the different treatments (n=3 replicates per sample) by measuring the amount of malondialdehyde (MDA) following the thiobarbituric acid (TBA) assay described by Heath & Packer (1968) and Catalá et al. (2010). We first prepared a 2 mM stock solution of the MDA precursor, malonaldehyde bis(diethyl acetal) (1,1,3,3-Tetraethoxypropane; Sigma Aldrich, T9889) and built standards of 0, 5, 10, 20, and 40 µM MDA by diluting the stock solution in 80% ethanol with 2% buthylated hydroytholuene (BHT; Fisher Scientific, ICN10116290). For each sample, we homogenized between 3.6 and 75.8 mg of frozen moss tissue in a tissue lyser (Qiagen TissueLyser II) during 2-4 min, in rounds of 30 sec. Samples were immersed in liquid N between rounds to prevent tissue melting. We then added 1 ml of 0.1% trichloroacetic acid (TCA; Fisher Scientific, ICN19605780) to each sample and standard, and vortexed them. We centrifuged all tubes at 10,000 g for 20 minutes and recovered 500 µl of supernatant. We added an equal volume of 20% TCA containing 0.5% TBA (Sigma Aldrich, T5500-25G) to each tube, followed by 5 µl of BHT. All tubes were incubated at 95ºC in a hot plate for 30 minutes, cooled quickly on ice, and centrifuged at 10,000 g for 15 min. Finally, we recovered the supernatant, and measured its absorbance at 532 and 600 nm in a 96-well spectrophotometric microplate reader (Epoch Biotek). The concentration of MDA was calculated after subtracting the absorbance at 600 nm from that at 532 nm to eliminate the possible interference of soluble sugars present in the samples (Du & Bramlage, 1992).