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.


Y(NO)
Non-regulated non-photochemical quenching of PS II Y(NPQ) Regulated non-photochemical quenching of PS II Hydrogen peroxide is produced in plants in a variety of metabolic and stress-inducible pathways 1 . The electron transport of chloroplasts and mitochondria as well as various peroxisomal and plasma-membrane localised oxidases produce superoxide anion radicals (O 2 ·− ), which are converted into H 2 O 2 by the superoxide-dismutase enzyme (SOD) [2][3][4] . Because H 2 O 2 is relatively stable in biological systems with a half-life of milliseconds to seconds 5 , it may act as a second messenger molecule or enzyme substrate [4][5][6] in addition to being a damaging oxidising agent when present in higher concentrations 7 . Pathogens and various abiotic factors were shown to increase H 2 O 2 production, although stress responses generally involve the activation of various H 2 O 2 neutralising enzymes as well. Elevated H 2 O 2 concentrations in plant leaves were documented in response to UV-B irradiation 8 , excess photosynthetically active radiation (PAR) 9 , high temperature 10-12 , drought 13 , or heavy metal stress 14 . Although H 2 O 2 was considered capable of diffusing across membranes by itself 15 , its transport between intracellular compartments and between cells is mainly through aquaporins 16 . Such mobility facilitates the molecule's messenger function but also requires the antioxidant control of local concentrations further away from H 2 O 2 production sites. Ultraviolet-exposed plants especially need to maintain an effective H 2 O 2 regulating system [17][18][19][20][21][22] since the UV-B (280-315 nm) component of sunlight not only elevates H 2 O 2 concentrations in planta but may also photoconvert H 2 O 2 to more hazardous hydroxyl radicals ( • OH) 8 . Earlier studies have shown that the proper activation of class-III plant peroxidase (POD) enzymes is a key factor in the successful acclimation to UV-B both in model plants exposed to supplemental UV radiation in growth chambers 20 and in sun leaves outdoors 23 . Our recent work with tobacco plants also showed that leaf acclimation to supplementary UV-B is realised through a selective activation of POD isoforms 22 .
There are numerous POD isoenzymes in a plant tissue, mainly in cell walls and vacuoles 24 , but phenolic peroxidases were also found in chloroplasts 25 . The common view is that POD enzymes are not substrate-selective but rather use a wide range of phenolic compounds as electron donors 26,27 depending on the availability of these secondary metabolites 28 . At least three distinct pathways have been identified to facilitate flavonoid transport among cellular locations 29,30 , and phenolic compounds were found in a variety of cell compartments including the cytosol, vacuole, ER, as well as chloroplast and nucleus 31,32 .
Acclimative responses to UV-B include an increase in leaf phenolic contents 33,34 and the biosynthesis of these secondary metabolites occurs under the regulation of UVR8, the UV-B photoreceptor 35 . Little is known about the molecular mechanism of UV-inducible peroxidase upregulation. UV-B-induced UVR8-regulated genes include a glutathione peroxidase in Arabidopsis thaliana 36 . However, to the best of our knowledge, UVR8-regulated POD genes have not been identified so far. The possibility of indirect ROS-mediated upregulation has been suggested as the mode of action of UV-B on genes encoding antioxidant enzymes 37 . Such signalling has been demonstrated to occur as an upregulating UV-B effect on the multi-function defence genes PR1 and PDFI.2 38 . This model is supported by the overlap between antioxidant responses to UV-B and several other abiotic factors 7 . Given the well-established role of H 2 O 2 as a signal molecule, a plausible assumption is that UV-B stimulates leaf antioxidants through the increased production of ROS. In order to test this hypothesis, here, we compare the antioxidant responses of tobacco (Nicotiana tabacum) leaves to supplementary UV radiation and to direct H 2 O 2 treatment. The latter was achieved as irrigation with a water solution of H 2 O 2 , which has already been shown to increase H 2 O 2 concentrations in an experiment performed with rice seedlings 39 . The main phenolics in tobacco leaves include chlorogenic acid, rutin, and caffeic acid, but the presence of vanillic acid, ferulic acid, or quercetin has also been reported [40][41][42][43][44][45] .
The second aim of our work was to study whether UV-inducible phenolic compounds contribute to modulating leaf H 2 O 2 concentrations as POD substrates or as direct ROS scavengers. The latter function is feasible because several phenolic compounds are highly reactive to H 2 O 2 in vitro 46 . Regardless of their mode of action, phenolic compounds are oxidised when acting as antioxidants. This yields a wide range of products, which include phenoxyl and semiquinone radicals 47,48 . The chemistry of these reactions has been studied extensively in nutrition science 49 and results may also be relevant to reactions assumed to occur in planta. However, the threat of antioxidant phenolic compounds turning pro-oxidant is less likely in plant than in animal tissues. Experiments using a specific class-III plant peroxidase, horseradish peroxidase (HRP), demonstrated that phenolic antioxidants can be regenerated from their radical forms by ascorbate (ASA) 50 , glutathione (GSH) 51 , or by monodehydroascorbate reductase 52 .
As exposure to UV-B radiation affects the way plants respond to changes in other environmental factors 53 , our experimental setup also provided an opportunity to test the following hypothesis: Do responses to exogenous H 2 O 2 and endogenous, UV-B triggered H 2 O 2 overlap? To this end, a two-factor treatment, H 2 O 2 irrigation under supplemental UV-B, was also added.

Results
Tobacco leaves were analysed from plants in four treatment groups: (1) kept under PAR only and irrigated with water (untreated control, C), (2) Figure S1). In fact, a slight (6-8%) increase in yield was detected in treated plants compared to untreated controls. This observation shows that leaves were not damaged by but are rather acclimated to the applied treatments. www.nature.com/scientificreports/ UV-B irradiation and H 2 o 2 treatment induce distinct antioxidant responses. Leaf H 2 O 2 contents were significantly increased by both treatments, and the effects of these were interactive: one factor had a stronger positive effect in the presence of the other (Fig. 1A). SOD activity was lower both in UV-B-exposed and in H 2 O 2 -treated plants than in untreated controls, and the interaction of the two factors was negative (Fig. 1B). APX activity was increased by ca. 50% in UV-B treated leaves but was unaffected by the H 2 O 2 treatment either in the presence or absence of UV-B (Fig. 1C). When GPX was assayed using H 2 O 2 as substrate, the enzyme activity showed only a non-significant marginal (p = 0.084) increase in UV-B treated leaves. Plant H 2 O 2 treatment had no effect on enzyme activity (Fig. 1D). Using an organic hydroperoxide as GPX substrate revealed no differences in the activities as well (data not shown). The effects of UV-B and H 2 O 2 on CAT activity were opposed: UV-B had a positive (ca. 47%) but H 2 O 2 treatment had a negative (ca. − 87%) effect. Despite its negative effect as a single factor, the H 2 O 2 treatment had no effect on how CAT activity reacted to UV-B (Fig. 1E). Non-enzymatic H 2 O 2 neutralising capacities showed relatively small changes in response to the applied treatments. H 2 O 2 treatment as a single factor resulted in a ca. 20% higher antioxidant capacity and UV-B alone had no significant effect. However, the two factors interacted and the H 2 O 2 treatment resulted in a larger ca. 35% increase when UV-B was also applied (Fig. 1F). Before examining POD responses, changes in leaf phenolics were also assessed. Dualex measurements indicated a strong (120-130%) increase in the adaxial phenolic index in response to the UV-treatment, both with and without the exogenous H 2 O 2 treatment. The application of H 2 O 2 alone had no significant effect (data not shown). Because the Dualex technique is based on 375 nm absorption in leaf tissue 54 , it is expected to reflect an increase in flavonoids to a larger extent than those in phenolic acid content due to differences between the UV absorption of these two compound groups 46 . TLC separation of methanolic leaf extracts was attempted to illustrate changes in phenolic compounds (Supplementary Figure S2). However, base levels in untreated leaves were too low for detection. As expected from Dualex measurements, the H 2 O 2 treatment alone did not amend this situation. Extracts from UV-B exposed leaves, however, showed a large increase in chlorogenic acid (CGA) and the flavonol quercetin-rutinoside (RUT) contents. Since the TLC technique does not allow quantitative comparisons, our results only indicate that this marked change in phenolic composition was a common characteristic of UV-B and UV-B + H 2 O 2 treated leaves (Supplementary Figure S2).
UV-B responsive phenolic compounds support diverse defence functions. In order to investigate the possible contributions of the two major UV-B responsive phenolic compounds RUT and CGA to the nonenzymatic H 2 O 2 neutralising capacity of leaves, we used pure test compounds. This is an extension of a previous study, which reported the non-enzymatic H 2 O 2 neutralising capacities of 36 different phenolic compounds including RUT and QUE but not CGA 46 . In addition to phenolic compounds used as POD substrates in the present study, two non-phenolic antioxidants, ASA and GSH, were also added due to their potential to re-reduce oxidised phenolics 50,51 . The results are shown in Table 1, relative to the non-enzymatic H 2 O 2 neutralising capacity of ASA. The two phenolic acids, CGA and caffeic acid (CAA), were weaker antioxidants than ASA and much weaker than the two flavonols; their H 2 O 2 neutralising capacities were similar to those of GSH in this in vitro model. Following this comparison of direct H 2 O 2 reactivities, we compared phenolic compounds as POD substrates.
Using ABTS or guaiacol as substrates is a common practice when POD activities are assayed. We complemented these two methods by using four different phenolic compounds that occur in tobacco leaves. CGA and RUT were chosen because these were present in higher amounts in UV-B treated leaves ( Supplementary Figure S2). The choice of the aglycone form of RUT (quercetin, QUE) was based on the earlier use of this substrate to assess POD in tobacco leaves 22 . CAA was included in the study as a reported major phenolic component of tobacco leaves 55 . In the following, POD activities are discussed according to the substrate used in the assay; for example, RUT-POD refers to POD activity measured using RUT as substrate. Figure 2 shows that the results were strongly affected by the choice of substrate, both in activity (enzyme units) and in terms of responses to treatments. POD enzyme units measured in untreated leaves using various substrates followed a RUT-POD > CGA-POD > ABTS-POD > CAA-POD > Gua-POD > QUE-POD > order. Activities measured in untreated leaves with these substrates are given in legends to Figs.1 and 2 in enzyme units. CAA-POD and RUT-POD showed no change in response to either treatment (Fig. 2D,F, respectively). ABTS-POD increased in response to UV-B and H 2 O 2 treatment by ca. 120% and 30%, respectively, compared to untreated leaves. The positive effect of UV-B was maintained in H 2 O 2 treated plants, although the two factors interacted and the H 2 O 2 treatment lessened the extent of the positive UV-B effect ( Fig. 2A). Gua-POD and CGA-POD was increased by UV-B regardless of the application of H 2 O 2 treatment, which was not a significant factor. Interestingly, the two treatments had opposite effects on QUE-POD: the UV-B treatment increased the enzyme activity, while the H 2 O 2 treatment decreased it. These effects were maintained as significant in the two factor treatment without interaction, and the positive effect of UV-B was smaller without the H 2 O 2 treatment (Fig. 2C).
In the following section, we studied the effects of ASA or GSH on the oxidation rates of QUE, RUT, CAA, or CGA as POD substrates. In this experiment, various amounts of ASA or GSH were added and the kinetics of phenolic substrate oxidation by POD enzymes contained in the leaf extract was followed photometrically at the indicated wavelengths. ASA and GSH were used in the 1.4 to 140 μM concentration range and the ability of the reactivity of these antioxidants to restore oxidised phenolic substrates was illustrated by a time delay in the consumption of these compounds. In this experiment, we used a pooled sample of UV-B treated leaves because these had the highest relative POD activities (Fig. 2). The oxidation rate of RUT was not affected by the presence of either ASA or GSH up to 140 μM concentrations (data not shown). Figure 3 shows that GSH was most reactive to oxidised CGA (Fig. 3A), less reactive to oxidised CAA (Fig. 3B), and did not restore oxidised QUE, even www.nature.com/scientificreports/ at the highest applied concentration (Fig. 3C). The efficiency of the same concentration (20 μM) ASA to retard phenolic substrate oxidation followed an opposite QUE > CAA > CGA order (Fig. 3).

Discussion
Using model plants in growth chambers, the present work shows that UV-B irradiation selectively enhances a subset of the antioxidant network. Analysing data from publications, which reported UV-induced changes in antioxidant enzyme activities, we have already shown that the stronger activation of peroxidases than SOD is a special characteristic of acclimative plant responses to UV-B 20 . The role of this response is to avoid high leaf H 2 O 2 levels prone to UV-B photocleavage into hydroxyl radicals 8 . The present study supports this model. Moreover, it shows that successful acclimation (avoided loss in leaf photochemical yield) may also be realised with a decrease in SOD activity combined with increased APX, POD, and CAT activities (Figs. 1 and 2). In the present experiment, GPX activities were not affected by the applied UV-B treatment (Fig. 1D), contrary to earlier reports on UV-B regulated GPX expression in Arabidopsis 56 . This may be due to differences in the UV-B fluence rates applied and in the levels of detection (gene vs. enzyme activity). CAT activities were significantly lower (20.6 and 30.1 mU mg protein −1 control and in UV-B acclimated leaves, respectively) than those of APX (347 and 516 mU mg protein −1 ) or POD (1-100 U mg protein −1 , depending on substrate and treatment), which is probably due to the low photorespiration in leaves grown under relatively low PAR in this experiment. POD responses to UV-B showed an interesting heterogeneity and suggest that only specific isoforms contribute to the acclimation. Our earlier study has already demonstrated that two POD assays using traditional substrates, ABTS and guaiacol, registered different extents of POD activation in UV-B-treated tobacco leaves 22 . Using four phenolic compounds naturally occurring in tobacco leaves (CAA, CGA, RUT, QUE), we show here that although these can be oxidised as POD substrates, only specific POD isoforms contribute to acclimation to supplemental UV-B (Fig. 2). This conclusion is similar, but not identical, to that of Jansen et al. 57 . Studying the UV-susceptibility of transgenic tobacco lines over-expressing phenol-oxidising peroxidases, the authors put forward a model in which isoenzyme diversity resulted in the polymerisation and/or crosslinking of specific phenolic compounds, and which increased the protection of plants from UV-radiation 57 . While not debating the validity of their model, we offer an alternative, which can be coexisting in leaves. Our hypothesis is based on the assumptions that (1) phenolic compounds protect leaves from radiation not only as UV screening compounds but also as antioxidants, (2) phenolics oxidatively modified by either POD or in a direct reaction with H 2 O 2 can be re-generated, and (3) the assignment of individual compounds to defence functions depends not only on their antioxidant capacities but also on the metabolic economy of their regeneration. Several authors have already shown that the phenolic compounds included in our study act as electron donors to a specific POD form: HRP 58,59 ). Our data show that these are also capable of supporting other POD, such as the ones in tobacco leaves, although the biochemical properties of the tobacco enzyme were found to be distinct from those of HRP 60 . First, we discuss the possible roles of the two phenolic components abundant in UV acclimated leaves, CGA and RUT. Even in the absence of the UV treatment, both compounds were efficient POD substrates, conferring 15 to 90-times higher POD activities than CAA or QUE in untreated leaves. The complexity of the phenolic defence response is supported by the result that substrates favoured by UV-responsive POD isoforms (CGA and QUE, Fig. 2) do not fully correspond to compounds accumulated in high amounts in UV-treated leaves (CGA and RUT, Supplementary Fig. 2). However, while CGA-POD was ca. 50% more active in UV-B exposed leaves than in controls, there was no significant change in RUT-POD (Fig. 2E,D, respectively). On the other hand, RUT is a strong direct H 2 O 2 neutralising antioxidant, with 1.65-times higher reactivity to ROS than ASA (Table 1). Therefore, the explanation for the substantial increase in leaf RUT content in UV-exposed leaves is not the increased need for this compound as electron donor to POD but rather as a direct antioxidant. Contrary to RUT, CGA is a relatively poor non-enzymatic antioxidant (Table 1) but an efficient POD substrate. POD-oxidised CGA was efficiently recovered by ASA or GSH. The former finding is in agreement with earlier reports using HRP 58,61 , and the latter is a novel one. We found no such recovery in the case of POD-oxidised RUT. This difference also supports the participation of CGA-POD, but not RUT-POD, in the observed UV response. CGA biosynthesis is reportedly induced by a variety of stress conditions in addition to UV 62 , and our results suggest that its main role is lessening damage as a POD substrate.
Considering the other two phenolic leaf components, CAA and QUE, the amounts of these were either unaffected by the UV exposure or the increase was minor and below the detection threshold of the applied TLC method. CAA is a relatively weak direct antioxidant (Table 1) and the relatively low POD activity it conferred (6% of CGA-POD) did not change upon UV exposure (Fig. 2F). These observations suggest that the role of CAA in the UV response is negligible. QUE, an aglycone flavonol, is of more interest. First, flavonoid aglycones are www.nature.com/scientificreports/ www.nature.com/scientificreports/ usually present in leaves in much lower amounts than their glycosylated forms, such as RUT 63 . Thus, the low activity of QUE-POD (ca. 90-times lower than RUT-POD and 45-times lower than CGA-POD in untreated leaves) may be explained by a relatively low amount of POD isoforms preferring a substrate in short supply. However, the significance of QUE in the UV response should not be dismissed. QUE is a very efficient direct H 2 O 2 antioxidant, 3.5-times stronger than ASA. Also, the relative activity of QUE-POD may be low, but it nearly doubled in response to UV-B. On the other hand, the regeneration of QUE from its oxidised form is the least 'economical' among the phenolic compounds in this study in the sense that it required more ASA than the restoration of CGA or CAA, and it was not recovered by GSH (Fig. 3). This result also suggests that the QUE form yielded by leaf QUE-POD is a phenoxyl radical rather than a GSH-reactive semiquinone, similar to the form identified in animal cells 51 .
In summary, the antioxidant aspect of tobacco leaf UV acclimation was realised mainly through lowered SOD and increased peroxidase activities; the latter involving isoforms that use CGA and, to a smaller extent, QUE as electron donors. Although the increased RUT pool indicates the potential of more efficient non-enzymatic H 2 O 2 neutralisation, there was no significant change in this function in UV exposed leaves (Fig. 1F), indicating that the contribution of RUT to this pathway was minor.
Nevertheless, H 2 O 2 levels were higher in these UV-B-acclimated leaves than in controls (Fig. 1A). A UVinducible increase in leaf H 2 O 2 concentrations has already been reported in stressed plants, where the irradiation resulted in a decrease in photosynthetic performance 8 but not yet in well-acclimated ones. Hydrogen peroxide is a well-established secondary messenger 1 and it is plausible that controlled low levels of this ROS participate in the induction of the antioxidant response to UV-B as well. Whether acclimative UV-responses are triggered   1 mM) was used for soil irrigation, and the treatment resulted in a 20% increase in leaf H 2 O 2 levels ( Fig. 1) but did not cause any loss in leaf photochemical yield. APX activity was unaffected, and the only positive effect on POD was detectable as ABTS-POD. None of the phenolic-substrate-using POD isoforms were stimulated by the H 2 O 2 (Fig. 2), confirming that the synthetic compound ABTS as electron donor assesses a different subset of leaf POD than natural compounds. Contrary to the UV-B treatment, which enhanced the enzymatic but not the non-enzymatic neutralisation of H 2 O 2 , the direct ROS treatment applied increased the latter defence pathway but had only a minor positive effect on enzymatic defence. Moreover, QUE-POD and CAT activities were lower (by 25% and 50%, respectively) in H 2 O 2 treated leaves than in controls. The only common response to the two different treatments was a decrease in SOD activity. This suggests that the source of neither UV-B-induced nor H 2 O 2 -irrigation-induced excess H 2 O 2 is an increased enzymatic conversion of superoxide radicals. Because exogenous H 2 O 2 resulted in an increase in non-enzymatic antioxidant capacity without a marked increase in phenolic content, the contribution of these compounds as direct H 2 O 2 scavengers was most likely minor in acclimating to this treatment. Factor interactions between UV-B and H 2 O 2 treatments were explored further using two-way ANOVA. Statistically verified interactions, which do not grant but only imply the possibility of crosstalk between the two factors, require two conditions: One is that single factors are significant in the two-factor experiment, for example, UV-B increasing the studied effect (e.g. enzyme activity) both in the absence and in the presence of exogenous H 2 O 2 (p < 0.05 in the first row of the inset tables in Figs. 1 and 2) and vice versa (p < 0.05 in the second row). The second condition is a p < 0.05 interaction (the third row in these tables). These two conditions are met only in the case of leaf H 2 O 2 content (Fig. 1A) and SOD (Fig. 1B). The effect of treatments was positive in the former and negative in the latter case. Lowering SOD-mediated production is possibly a common acclimative response to an increase in cellular H 2 O 2 levels, regardless of the nature of the external stimulus. A parallel application of UV-B and H 2 O 2 resulted in an additive effect on H 2 O 2 content in the sense that the simultaneous presence of the two factors led to an effect that was equal to the sum of the effects caused by the two factors applied separately 53 .
The major differences in antioxidant responses to H 2 O 2 and UV-B, as well as the very limited interaction between the two factors when applied in parallel, as discussed above, suggest that UV acclimation is unlikely to have been brought about by the UV-induced increase in leaf H 2 O 2 content. A difference in production H 2 O 2 sites in response to the two treatments may argue against this assumption, but the relatively long life-time and ability of this ROS to spread in tissues 16 diminishes the importance of this aspect. In the absence of evidence for UVR8-initiated activation of acclimative antioxidant signalling, one can only speculate on potential routes. If this pathway involves direct UV perception, then candidates include a UV-B photoreceptor distinct from UVR8 65 or a contribution of UV-A photoreceptors 64 as the broad-band UV source applied in our experiment contained UV-A as well. A metabolite initiated pathway may include oxidised ascorbate, which has already been implicated in responses to stressors other than UV radiation 66 or possibly, oxidised phenolic compounds.
Increased phenolic peroxidase activity has been widely reported as a general, non-specific defence response. Our present study, however, shows the existence of inducement-specific, phenolic substrate-dependent POD responses in UV-treated leaves and suggests a further investigation of the heterogeneity of POD responses under different abiotic stress conditions. Further, the present study also draws attention to the possibility of the novel yet unexplored complexity of POD responses to other stress conditions as well. As illustrated by the example of the UV-induced changes, an increase in a certain phenolic component in the leaf does not necessarily correspond to its increased use as POD substrate; thus, the latter cannot be fully explained by its increased availability. A more plausible model is the selective upregulation of POD isoforms using phenolic substrates, which can be recovered from their oxidised form by relatively low amounts of other antioxidants, such as ascorbate or glutathione. This hypothesis is supported by our data but must be verified further through a quantitative analysis of antioxidant metabolites and correlations between changes in their levels during UV acclimation. . Enzyme activities were quantified following the oxidation of the corresponding substrate as absorption change using a spectrophotometer (Shimadzu UV1800, Shimadzu Corp., Kyoto, Japan). Substrate concentrations in the reaction mixture and absorbance wavelengths are summarised in Table 2 along with the molar extinction coefficients used to calculate enzyme activities as mU activity mg −1 protein, where 1 U = 1 mM substrate min −1 . References describing the details of original methods are also listed in Table 2. When indicated, the reaction mixture contained either ascorbate (7-14 µM) or GSH (1.4-140 µM) in addition to one of the phenolic compounds.
Ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured according to Nakano and Asada 75 by following the oxidation of ascorbate at 295 nm in a sodium phosphate buffer (50 mM, pH 7.0) containing 1 mM Table 2. Characterization of POD enzyme substrates used in the present study. *Due to the fast oxidation of quercetin a substrate, the reaction was followed by the loss of ascorbate due to recovering oxidized quercetin at 295 nm, using ε 295 nm = 1.47 mM −1 cm −1 . www.nature.com/scientificreports/ EDTA, 0.5 mM ascorbate, and 1 mM H 2 O 2 . The results were corrected for APX-independent H 2 O 2 reduction, which was typically less than 10% of enzymatic rates. Enzyme activities were calculated using the molar extinction coefficient of ascorbate (ε 295 nm = 1.47 mM −1 cm −1 ) as mU APX mg −1 protein.
Catalase (CAT, EC 1.11.1.6) activity was determined as described by Aebi et al. 77 by following the decrease in H 2 O 2 concentration as 240 nm absorbance in a reaction mixture containing 18.6 mM H 2 O 2 and 1 mM EDTA in a 50 mM sodium-phosphate buffer (pH 7.0). The reaction was started by adding 60 µL leaf sample (corresponding to 1.3-4.1 µg soluble protein) and CAT activities were given as mU mg −1 protein.
non-enzymatic H 2 o 2 antioxidant capacity measurement. Hydrogen peroxide neutralising antioxidant capacities were evaluated through the photometric detection of iodine (I 2 ) yielded in the reaction between H 2 O 2 and potassium iodide (KI), and the ability of H 2 O 2 reactive compounds to lessen the amount of this product 78 . For this experiment frozen leaves were powdered in liquid N 2 using a pestle and mortar and extracted in 70% (v/v) ethanol. The reaction mixture contained 25 μM H 2 O 2 , 595 μM KI in potassium-phosphate buffer (pH 7.0) and either leaf extracts (corresponding to 300 μg leaf FW) or one of the pure test compounds (0.42-3.2 mM). The final concentration of ethanol in the reaction mixture was always 7.5% (v/v). Absorption at 405 nm was measured twice, immediately and 3 min after mixing assay components using a Multiskan FC plate reader (Thermo Fischer Scientific, Shanghai, China). Non-enzymatic H 2 O 2 antioxidant capacities were given as μM ascorbic acid (ASA) equivalents.
Hydrogen peroxide content measurement. Leaf H 2 O 2 levels were estimated using a photometric assay 79 based on the H 2 O 2 -induced absorption change of 125 μM xylenol orange in 6% (v/v) trichloroacetic acid (TCA). For this assay, samples were collected from plants within the growth chamber under light conditions corresponding to treatment groups i.e. PAR only or PAR plus UV-B. Three leaf disks corresponding to 26-56 mg FW were homogenised in 6% TCA immediately after cutting, centrifuged (15,000×g, 10 min, 4 °C, Heraeus Fresco 17 Centrifuge, Thermo Fisher Scientific, Waltham, USA), and the supernatants were incubated for 30 min before detecting 560 nm absorptions. Leaf H 2 O 2 contents were given in nM mg −1 FW units using calibration curves in the 0-10 nM H 2 O 2 range. Statistical analysis. Each treatment group contained four plants. One leaf from each plant was chosen for the analyses, and all measurements were performed in 3-4 repetitions. Results are presented as means ± standard deviations. The combined and single factor effects of UV-B and H 2 O 2 were analysed with a two-way ANOVA. Three null hypotheses were tested: (1) the H 2 O 2 treatment had no effect, (2) the absence/presence of UV-B over the PAR background had no effect, and (3) there was no interaction between the two factors. Tukey HSD was used as post-hoc test and verified rejections of the ANOVA null hypotheses were characterised with p values. Statistical analyses were performed using the PAST software 80 .