Phenolic peroxidases: Dull generalists or purposeful specialists in stress responses?

This study focuses on class III peroxidases (POD) (EC 1.11.1.7) as regulators of cellular H2O2 levels in leaves under oxidative stress. The effective regulation of reactive oxygen species (ROS) concentrations in plant tissues is crucial for plant survival, and has been extensively reviewed. However, the majority of studies regard POD as a generalist without substrate specificity. This is partly due to the fact that laboratory protocols assessing POD levels use substrates, which are not contained in plants. Here, we show that both base- and stress-inducible POD activity depends on the choice of substrate. Moreover, the application of diverse substrates, particularly those contained in plants, unmasks POD isoenzymes that are distinguished by substrate preferences. This functional heterogeneity of POD responses is worth studying, especially in parallel with stress-induced changes in the phenolic profiles.

Narrow-Band 311 nm Ultraviolet-B Radiation Evokes Different Antioxidant Responses from Broad-Band Ultraviolet.

Supplemental narrow-band 311 nm UV-B radiation was applied in order to study the effect of this specific wavelength on tobacco as a model plant. UV-B at photon fluxes varying between 2.9 and 9.9 µmol m-2 s-1 was applied to supplement 150 µmol m-2 s-1 photosynthetically active radiation (PAR) for four hours in the middle of the light period for four days. Narrow-band UV-B increased leaf flavonoid and phenolic acid contents. In leaves exposed to 311 nm radiation, superoxide dismutase activity increased, but phenolic peroxidase activity decreased, and the changes were proportional to the UV flux. Ascorbate peroxidase activities were not significantly affected. Narrow-band UV-B caused a dose-dependent linear decrease in the quantum efficiency of photosystem II, up to approximately 10% loss. A parallel decrease in non-regulated non-photochemical quenching indicates potential electron transfer to oxygen in UV-treated leaves. In addition to a flux-dependent increase in the imbalance between enzymatic H2O2 production and neutralization, this resulted in an approximately 50% increase in leaf H2O2 content under 2.9-6 µmol m-2 s-1 UV-B. Leaf H2O2 decreased to control levels under higher UV-B fluxes due to the onset of increased non-enzymatic H2O2- and superoxide-neutralizing capacities, which were not observed under lower fluxes. These antioxidant responses to 311 nm UV-B were different from our previous findings in plants exposed to broad-band UV-B. The results suggest that signaling pathways activated by 311 nm radiation are distinct from those stimulated by other wavelengths and support the heterogeneous regulation of plant UV responses.

Ultraviolet-B acclimation is supported by functionally heterogeneous phenolic peroxidases.

Tobacco plants were grown in plant chambers for four weeks, then exposed to one of the following treatments for 4 days: (1) daily supplementary UV-B radiation corresponding to 6.9 kJ m-2 d-1 biologically effective dose (UV-B), (2) daily irrigation with 0.1 mM hydrogen peroxide, or (3) a parallel application of the two treatments (UV-B + H2O2). Neither the H2O2 nor the UV-B treatments were found to be damaging to leaf photosynthesis. Both single factor treatments increased leaf H2O2 contents but had distinct effects on various H2O2 neutralising mechanisms. Non-enzymatic H2O2 antioxidant capacities were increased by direct H2O2 treatment only, but not by UV-B. In contrast, enzymatic H2O2 neutralisation was mostly increased by UV-B, the responses showing an interesting diversity. When class-III peroxidase (POD) activity was assayed using an artificial substrate (ABTS, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)), both treatments appeared to have a positive effect. However, only UV-B-treated leaves showed higher POD activities when phenolic compounds naturally occurring in tobacco leaves (chlorogenic acid or quercetin) were used as substrates. These results demonstrate a substrate-dependent, functional heterogeneity in POD and further suggest that the selective activation of specific isoforms in UV-B acclimated leaves is not triggered by excess H2O2 in these leaves.

Selective responses of class III plant peroxidase isoforms to environmentally relevant UV-B doses.

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.