Efficient hydrogen peroxide detoxification is an essential aspect of plant defence against a large variety of stressors. Among others, class III peroxidase (POD, EC 1.11.1.7) enzymes provide this function. Previous studies have shown that PODs are present in several isoforms and have in general low substrate specificities. The aim of our work was to study how various assays based on using various substrates reflect differences in peroxidase activities of tobacco leaves due to either developmental or environmental factors. The former factor was studied comparing fully developed leaves of the 3rd and 5th nodes; and the latter was achieved using plants acclimated to low doses of supplementary UV-B (280-315 nm) in growth chambers. To investigate the above, POD activities were measured using three different, commonly used chromophore substrates: ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)), guaiacol (2-methoxyphenol), OPD (o-phenylenediamine) and a fourth substrate, the secondary metabolite quercetin. All substrates registered a UV-B induced increase in leaf peroxidases as compared to untreated controls, although to different extents. However, age-related differences between upper and lower leaves were only detectable when either ABTS or quercetin were used as substrates. Additionally, native PAGE separation of POD isoforms followed by visualisation using one of the substrates showed that leaf acclimation to supplementary UV-B is realized via a selective activation of POD isoforms.
Keywords: Leaf; Peroxidase; Substrate dependence; Tobacco; Ultraviolet light.
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