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.

Phase-Selective Synthesis of Anatase and Rutile TiO2 Nanocrystals and Their Impacts on Grapevine Leaves: Accumulation of Mineral Nutrients and Triggering the Plant Defense.

Titanium dioxide nanocrystals (TiO2 NCs), through their photocatalytic activity, are able to generate charge carriers and induce the formation of various reactive oxygen species (ROS) in the presence of O2 and H2O. This special feature makes TiO2 an important and promising material in several industrial applications. Under appropriate antioxidant balancing, the presence of ROS is crucial in plant growth and development, therefore, the regulated ROS production through the photocatalytic activity of TiO2 NCs may be also exploited in the agricultural sector. However, the effects of TiO2 NCs on plants are not fully understood and/or phase-pure TiO2 NCs are rarely used in plant experiments. In this work, we present a phase-selective synthesis of TiO2 NCs with anatase and rutile crystal phases. The nanomaterials obtained were characterized by means of X-ray diffraction (XRD), transmission electron microscopy (TEM), diffuse reflectance UV-Vis spectroscopy, and electron paramagnetic resonance spectroscopy (EPR). In field experiments, Vitis vinifera cv. Cabernet Sauvignon leaves developed under natural sunlight were treated with aqueous dispersions of TiO2 NCs at concentrations of 0.001, 0.01, 0.1, and 1 w/v%. The effect of the applied nanocrystals was characterized via leaf photochemistry, mineral nutrient contents, and pyridoxine levels. We found that stress responses of grapevine to anatase and rutile NCs treatments are different, which can be related to the different ROS profiles of the two polymorphs. Our results indicate that TiO2 NCs may be utilized not only for direct pathogen inactivation but also for eliciting plant defense mechanisms.

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.

Ultraviolet-B radiation exposure lowers the antioxidant capacity in the Arabidopsis thaliana pdx1.3-1 mutant and leads to glucosinolate biosynthesis alteration in both wild type and mutant.

Pyridoxine (vitamin B6) and its vitamers are used by living organisms both as enzymatic cofactors and as antioxidants. We used Arabidopsis pyridoxine biosynthesis mutant pdx1.3-1 to study the involvement of the PLP-synthase main polypeptide PDX1 in plant responses to ultraviolet radiation of two different qualities, one containing primarily UV-A (315-400 nm) and the other containing both UV-A and UV-B (280-315 nm). The antioxidant capacity and the flavonoid and glucosinolate (GS) profiles were examined. As an indicator of stress, Fv/Fm of photosystem II reaction centers was used. In pdx1.3-1, UV-A + B exposure led to a significant 5% decrease in Fv/Fm on the last day (day 15), indicating mild stress at this time point. The antioxidant capacity of Col-0 wildtype increased significantly (50-73%) after 1 and 3 days of UV-A + B. Instead, in pdx1.3-1, the antioxidant capacity significantly decreased by 44-52% over the same time period, proving the importance of a full complement of functional PDX1 genes for the detoxification of reactive oxygen species. There were no significant changes in the flavonoid glycoside profile under any light condition. However, the GS profile was significantly altered, both with respect to Arabidopsis accession and exposure to UV. The difference in flavonoid and GS profiles reflects that the GS biosynthesis pathway contains at least one pyridoxine-dependent enzyme, whereas no such enzyme is used in flavonoid biosynthesis. Also, there was strong correlation between the antioxidant capacity and the content of some GS compounds. Our results show that vitamin B6 vitamers, functioning both as antioxidants and co-factors, are of importance for the physiological fitness of plants.

Multiple roles for Vitamin B6 in plant acclimation to UV-B.

Direct and indirect roles of vitamin B6 in leaf acclimation to supplementary UV-B radiation are shown in vitamin B6 deficient Arabidopsis thaliana mutant rsr4-1 and C24 wild type. Responses to 4 days of 3.9 kJ m-2 d-1 biologically effective UV-B dose were compared in terms of leaf photochemistry, vitamer content, and antioxidant enzyme activities; complemented with a comprehensive study of vitamer ROS scavenging capacities. Under UV-B, rsr4-1 leaves lost more (34%) photochemical yield than C24 plants (24%). In the absence of UV-B, rsr4-1 leaves contained markedly less pyridoxal-5'-phosphate (PLP) than C24 ones, but levels increased up to the C24 contents in response to UV-B. Activities of class-III ascorbate and glutathione peroxidases increased in C24 leaves upon the UV-B treatment but not in the rsr4-1 mutant. SOD activities remained the same in C24 but decreased by more than 50% in rsr4-1 under UV-B. Although PLP was shown to be an excellent antioxidant in vitro, our results suggest that the UV-B protective role of B6 vitamers is realized indirectly, via supporting peroxidase defence rather than by direct ROS scavenging. We hypothesize that the two defence pathways are linked through the PLP-dependent biosynthesis of cystein and heme, affecting peroxidases.

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.

Elevated ROS-scavenging enzymes contribute to acclimation to UV-B exposure in transplastomic tobacco plants, reducing the role of plastid peroxidases.

Leaf peroxidases play a key role in the successful acclimation of plants to low UV-B doses. The aim of the present study was to examine whether selective enhancement of alternative chloroplast antioxidant pathways achieved by chloroplast transformation affected the need for peroxidase defense. Transplastomic tobacco lines expressing glutathione reductase in combination with either dehydroascorbate reductase or glutathione-S-transferase in their plastids exhibited better tolerance to supplemental UV-B than wild type plants. After 10days UV treatment, both the maximum and effective quantum yields of PSII decreased in the wild type by 10% but were unaffected in either of the transformed lines. Activities of total peroxidase and ascorbate peroxidase, in addition to dehydroascorbate reductase and gluthatione-S-transferase, were increased by UV in all lines. Gluthatione reductase activity was unaffected by UV in the transplastomic line engineered to have a higher constitutive level of this enzyme, but increased in the two other genotypes. However, the observed more successful acclimation required less activation of peroxidases in the doubly transformed plants than in the wild type and less increase in non-enzymatic hydroxyl radical neutralization in the dehydroascorbate reductase plus glutathione reductase fortified plants than in either of the other lines. These results highlight the fundamental role of efficient glutathione, and especially ascorbate, recycling in the chloroplast in response to exposure of plants to UV-B. They also identify chloroplast localized peroxidases among the large variety of leaf peroxidases as essential elements of defense, supporting our earlier hypothesis on hydrogen peroxide UV-B photo-cleavage as the primary mechanism behind damage.

Light piping driven photosynthesis in the soil: Low-light adapted active photosynthetic apparatus in the under-soil hypocotyl segments of bean (Phaseolus vulgaris).

Photosynthetic activity was identified in the under-soil hypocotyl part of 14-day-old soil-grown bean plants (Phaseolus vulgaris L. cv. Magnum) cultivated in pots under natural light-dark cycles. Electron microscopic, proteomic and fluorescence kinetic and imaging methods were used to study the photosynthetic apparatus and its activity. Under-soil shoots at 0-2cm soil depth featured chloroplasts with low grana and starch grains and with pigment-protein compositions similar to those of the above-soil green shoot parts. However, the relative amounts of photosystem II (PSII) supercomplexes were higher; in addition a PIP-type aquaporin protein was identified in the under-soil thylakoids. Chlorophyll-a fluorescence induction measurements showed that the above- and under-soil hypocotyl segments had similar photochemical yields at low (10-55µmolphotonsm(-2)s(-1)) light intensities. However, at higher photon flux densities the electron transport rate decreased in the under-soil shoot parts due to inactivation of the PSII reaction centers. These properties show the development of a low-light adapted photosynthetic apparatus driven by light piping of the above-soil shoot. The results of this paper demonstrate that the classic model assigning source and sink functions to above- and under-soil tissues is to be refined, and a low-light adapted photosynthetic apparatus in under-soil bean hypocotyls is capable of contributing to its own carbon supply.

UV-B effects on leaves-Oxidative stress and acclimation in controlled environments.

As the steady decline in the Earth's stratospheric ozone layer and parallel increase in solar ultraviolet-B (UV-B: 280-315nm) has come to an end, the focus of plant UV research has been shifted from regarding UV-B as threatening plant life to recognizing it as a regulatory factor. While UV-B photoreceptor mediated signaling is increasingly understood, the role of UV-B inducible reactive oxygen species is still to be explored. Earlier experiments with high UV-B irradiation doses and isolated thylakoid membranes demonstrated the potential of UV-B to trigger oxidative stress. However, under realistic UV conditions pro-oxidants cannot be reliably traced in more complex biological samples possessing an array of antioxidant defenses. In the absence of direct experimental evidence we must rely on indications and propose hypotheses on how and whether pro-oxidants, such as reactive oxygen species contribute to acclimative responses. Here we briefly review how a balance between pro-oxidants and antioxidants is affected by UV-B in whole plant experiments performed in controlled environments. A working hypothesis is proposed in which the extents of UV-induced peroxidase and superoxide dismutase activations affect the success of acclimation to UV-B.

Antioxidant defence in UV-irradiated tobacco leaves is centred on hydrogen-peroxide neutralization.

Greenhouse grown tobacco (Nicotiana tabacum L. cv. Petit Havana) plants were exposed to supplemental UV centred at 318 nm and corresponding to 13.6 kJ m(-2) d(-1) biologically effective UV-B (280-315 nm) radiation. After 6 days this treatment decreased photosynthesis by 30%. Leaves responded by a large increase in UV-absorbing pigment content and antioxidant capacities. UV-stimulated defence against ROS was strongest in chloroplasts, since activities of plastid enzymes FeSOD and APX had larger relative increases than other, non-plastid specific SODs or peroxidases. In addition, non-enzymatic defence against hydroxyl radicals was doubled in UV treated leaves as compared to controls. In UV treated leaves, the extent of activation of ROS neutralizing capacities followed a peroxidases > hydroxyl-radical neutralization > SOD order. These results suggest that highly effective hydrogen peroxide neutralization is the focal point of surviving UV-inducible oxidative stress and argue against a direct signalling role of hydrogen peroxide in maintaining adaptation to UV, at least in laboratory experiments.

Hydrogen peroxide contributes to the ultraviolet-B (280-315 nm) induced oxidative stress of plant leaves through multiple pathways.

Solar UV-B (280-315 nm) radiation is a developmental signal in plants but may also cause oxidative stress when combined with other environmental factors. Using computer modeling and in solution experiments we show that UV-B is capable of photosensitizing hydroxyl radical production from hydrogen peroxide. We present evidence that the oxidative effect of UV-B in leaves is at least twofold: (i) it increases cellular hydrogen peroxide concentrations, to a larger extent in pyridoxine antioxidant mutant pdx1.3-1 Arabidopsis and; (ii) is capable of a partial photo-conversion of both 'natural' and 'extra' hydrogen peroxide to hydroxyl radicals. As stress conditions other than UV can increase cellular hydrogen peroxide levels, synergistic deleterious effects of various stresses may be expected already under ambient solar UV-B.