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Towards understanding vacuolar antioxidant mechanisms: a role for fructans?

Recent in vitro, in vivo, and theoretical experiments strongly suggest that sugar-(like) molecules counteract oxidative stress by acting as genuine reactive oxygen species (ROS) scavengers. A concept was proposed to include the vacuole as a part of the cellular antioxidant network. According to this view, sugars and sugar-like vacuolar compounds work in concert with vacuolar phenolic compounds and the ‘classic’ cytosolic antioxidant mechanisms. Among the biologically relevant ROS (H2O2, O2·–, and ·OH), hydroxyl radicals are the most reactive and dangerous species since there are no enzymatic systems known to neutralize them in any living beings. Therefore, it is important to study in more detail the radical reactions between ·OH and different biomolecules, including sugars. Here, Fenton reactions were used to compare the ·OH-scavenging capacities of a range of natural vacuolar compounds to establish relationships between antioxidant capacity and chemical structure and to unravel the mechanisms of ·OH–carbohydrate reactions. The in vitro work on the ·OH-scavenging capacity of sugars and phenolic compounds revealed a correlation between structure and ·OH-scavenging capacity. The number and position of the C=C type of linkages in phenolic compounds greatly influence antioxidant properties. Importantly, the splitting of disaccharides and oligosaccharides emerged as a predominant outcome of the ·OH–carbohydrate interaction. Moreover, non-enzymatic synthesis of new fructan oligosaccharides was found starting from 1-kestotriose. Based on these and previous findings, a working model is proposed describing the putative radical reactions involving fructans and secondary metabolites at the inner side of the tonoplast and in the vacuolar lumen.

Comparative Evaluation of Total Antioxidant Capacities of Plant Polyphenols.

Thirty-seven samples of naturally occurring phenolic compounds were evaluated using three common in vitro assays for total antioxidant activity (TAC) testing: the Trolox Equivalent Antioxidant Capacity (TEAC), the Ferric Reducing Antioxidant Potential (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, in addition to the Folin-Ciocalteu reagent reactivity (FCR). We found that antioxidant hierarchies depended on the choice of assay and applied ANOVA analyses to explore underlying structure-TAC dependencies. In addition to statistically confirming the empirically established connection between flavonoid ring-B catechol and high TEAC or FRAP, new correlations were also found. In flavonoids, (i) hydroxyl groups on ring-B had a positive effect on all four TAC assays; (ii) the presence of a 3-hydroxyl group on ring-C increased TEAC and FRAP, but had no effect on DPPH or FCR; (iii) Phenolic acids lacking a 3-hydroxyl group had significantly lower FRAP or DPPH than compounds having this structure, while TEAC or FCR were not affected. Results demonstrated that any TAC-based ranking of phenolic rich samples would very much depend on the choice of assay, and argue for use of more than one technique. As an illustration, we compared results of the above four assays using either grapevine leaf extracts or synthetic mixtures of compounds prepared according to major polyphenols identified in the leaves.

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.

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

As the steady decline in the Earths 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.

Phenolic Compound Diversity Explored in the Context of Photo‐Oxidative Stress Protection

INTRODUCTION Phenolic compounds are a chemically diverse group of plant secondary metabolites with important roles both in plant stress defence and human nutrition. OBJECTIVE To explore structure-function relations potentiating phenolic compounds to promote leaf acclimation to light stress by excess photosynthetically active radiation (photoinhibition) and by solar ultraviolet (UV) radiation. METHODOLOGY We report singlet oxygen and hydrogen peroxide antioxidant capacities and UV-absorbing properties of 27 flavonoids and 11 phenolic acids. Correlations of these characteristics in the whole data set and related activity-structure relationships in flavonoid data were investigated using simple statistical methods. RESULTS In comparison to flavonoids, phenolic acids are relatively ineffective reactive oxygen neutralising antioxidants; and - with the exception of gallic acid - have poor reactivity to hydrogen peroxide. Singlet oxygen and hydrogen peroxide detoxifying capacities of flavonoids are positively correlated, largely due to the strong positive effect of the hydroxylation of the C-ring in position-3. 3-O-Glycosylation halves reactive oxygen species (ROS) reactivities of quercetin and myricetin but eradicates the hydrogen peroxide reactivity of kaemferol. B-ring polyhydroxylation (cathecol structure) increases the hydrogen peroxide antioxidant function but decreases UV-B (280-315 nm) absorption. UV-A (315-400 nm) absorption is increased by the B-ring C2-C3 double bond either in itself or in combination with the C4 oxo-group. CONCLUSION Among the studied compounds, anthocyanins and flavonols were the strongest singlet oxygen and hydrogen peroxide scavengers, and are thus capable of supporting defence against both photoinhibition by visible light and UV stress in leaves, while flavanols may only be effective against the latter. Copyright

Synthesis and potential use of 1,8-naphthalimide type 1O2 sensor molecules

New double (fluorescent and spin) sensor molecules containing 4-amino substituted 1,8-naphthalimide as a fluorophore and a sterically hindered amine (pre-nitroxide) or pyrroline nitroxide as a quencher and radical capturing moiety were synthesized. All sensors were substituted with a diethylaminoethyl side-chain to increase the water solubility. Steady state fluorescence properties of these compounds and their responses to ROS in vitro are reported with perspectives of plant physiology use in vivo.

Leaf hue measurements: A high-throughput screening of chlorophyll content

Computer analysis of digital photographic images provides fast, high-throughput screening of leaf pigmentation. Pixel-by-pixel conversion of red, green, blue (RGB) parameters to hue, saturation, value (HSV) showed that Hue values were proportional to total chlorophyll, offering an alternative to photometric analysis of leaf extracts. This is demonstrated using tobacco leaves with various chlorophyll contents due to senescence but shows the possibility of applications in studies of stress conditions accompanied by chlorophyll loss.

Environmental plasticity of Pinot noir grapevine leaves: A trans-European study of morphological and biochemical changes along a 1,500-km latitudinal climatic gradient

A 2-year study explored metabolic and phenotypic plasticity of sun-acclimated Vitis vinifera cv. Pinot noir leaves collected from 12 locations across a 36.69-49.98°N latitudinal gradient. Leaf morphological and biochemical parameters were analysed in the context of meteorological parameters and the latitudinal gradient. We found that leaf fresh weight and area were negatively correlated with both global and ultraviolet (UV) radiation, cumulated global radiation being a stronger correlator. Cumulative UV radiation (sumUVR) was the strongest correlator with most leaf metabolites and pigments. Leaf UV-absorbing pigments, total antioxidant capacities, and phenolic compounds increased with increasing sumUVR, whereas total carotenoids and xanthophylls decreased. Despite of this reallocation of metabolic resources from carotenoids to phenolics, an increase in xanthophyll-cycle pigments (the sum of the amounts of three xanthophylls: violaxanthin, antheraxanthin, and zeaxanthin) with increasing sumUVR indicates active, dynamic protection for the photosynthetic apparatus. In addition, increased amounts of flavonoids (quercetin glycosides) and constitutive β-carotene and α-tocopherol pools provide antioxidant protection against reactive oxygen species. However, rather than a continuum of plant acclimation responses, principal component analysis indicates clusters of metabolic states across the explored 1,500-km-long latitudinal gradient. This study emphasizes the physiological component of plant responses to latitudinal gradients and reveals the physiological plasticity that may act to complement genetic adaptations.

A comparison of colorimetric assays detecting hydrogen peroxide in leaf extracts

Hydrogen peroxide (H2O2) is a central molecule in plant stress responses as a potential oxidizing agent or a signal molecule, depending on its localization and cellular concentrations. The present work compares three colorimetric assays assessing H2O2 concentrations in terms of sensitivity, linearity with increasing H2O2 concentrations, solvent interactions and recovery of exogenous H2O2 from leaf extracts. The tested methods were based on the oxidation of one of the following chromophores: potassium iodide (KI), 3,3′-diaminobenzidine (DAB) or xylenol orange (XO). Various solvents were chosen for extraction to represent two different approaches of sampling: extraction into water or water-based buffer providing material for an array of bioassays and extraction into solutions containing trichloroacetic acid (TCA) or ethanol in order to minimize interactions arising from leaf biochemistry. The most employable technique was the xylenol orange assay in organic solvents, although it is advisable to interpret results against a series of spiked samples rather than a calibration line acquired in solvents only.

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.