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.

A novel procedure to assess the non-enzymatic hydrogen-peroxide antioxidant capacity of metabolites with high UV absorption.

Assays assessing non-enzymatic hydrogen peroxide antioxidant capacities are often hampered by the high UV absorption of the sample itself. This is a typical problem in studies using plant extracts with high polyphenol content. Our assay is based on comparing the 405 nm absorption of the product of potassium iodine and hydrogen peroxide in the presence and absence of a putative hydrogen peroxide reactive antioxidant. This method is free of interference with either hydrogen peroxide or antioxidant self-absorption and it is also suitable for high-throughput plate reader applications.

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.

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.

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 (H(2)O(2), O(2)·(-), 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.

The food additives inulin and stevioside counteract oxidative stress.

Prebiotics such as inulin (Inu)-type fructans and alternative natural sweeteners such as stevioside (Ste) become more popular as food ingredients. Evidence is accumulating that carbohydrates and carbohydrate-containing biomolecules can be considered true antioxidants, capable of scavenging reactive oxygen species (ROS). Here, we report on the ROS scavenging abilities of Inu and Ste in comparison with other sugars, sugar derivatives and arbutin. It is found that Inu and Ste are superior scavengers of both hydroxyl and superoxide radicals, more effective than mannitol and sucrose. Other compounds, such as 1-kestotriose, trehalose, raffinose and L-malic acid, also showed good reactivity to at least one of the two oxygen free radicals. The strong antioxidant properties of Inu and Ste are discussed. Within the plant vacuole, these compounds could play a crucial role in antioxidant defense mechanisms to help survive stresses. Addition to food assists in natural sweetening, food stabilization and maximizes health impact.