UV-irradiation provokes generation of superoxide on cell wall polygalacturonic acid.

We examined the redox effects of UV irradiation on cell wall isolates from Pisum sativum leaves, and polygalacturonic and galacturonic acid, in the presence of hydrogen peroxide. For this purpose, electron paramagnetic resonance spectroscopy and two spin-traps (DEPMPO and BMPO), capable of differentiating between various free radicals, were applied. Systems were exposed to UV-B (maximum emission at 312 nm) and UV-A (352 nm) for 10 min (6 J m(-2) s(-1)). Cell wall isolates exposed to UV in the presence of hydrogen peroxide, produced hydroxyl radical, carbon dioxide radical and superoxide. The production of superoxide was observed for cell wall isolates, polygalacturonic acid (in the presence and in the absence of calcium) and galacturonic acid, and it was diminished upon superoxide dismutase supplementation. The production is at least partially based on the reaction of hydroxyl radicals with (poly)galacturonic acid having carbon dioxide radicals as a products. Acting as a strong reducing agent, carbon dioxide radical reacts with molecular oxygen to produce superoxide. The results presented here shed a new light on: (1) the redox-modulating role of cell wall; (2) the production of superoxide in the extracellular compartment; (3) the mechanisms involved in translating UV stress into molecular signaling and (4) some other UV-related phenomena in plants, such as CO(2) emission.

Desiccation Tolerance in Ramonda serbica Panc.: An Integrative Transcriptomic, Proteomic, Metabolite and Photosynthetic Study.

The resurrection plant Ramonda serbica Panc. survives long desiccation periods and fully recovers metabolic functions within one day upon watering. This study aimed to identify key candidates and pathways involved in desiccation tolerance in R. serbica. We combined differential transcriptomics and proteomics, phenolic and sugar analysis, FTIR analysis of the cell wall polymers, and detailed analysis of the photosynthetic electron transport (PET) chain. The proteomic analysis allowed the relative quantification of 1192 different protein groups, of which 408 were differentially abundant between hydrated (HL) and desiccated leaves (DL). Almost all differentially abundant proteins related to photosynthetic processes were less abundant, while chlorophyll fluorescence measurements implied shifting from linear PET to cyclic electron transport (CET). The levels of H2O2 scavenging enzymes, ascorbate-glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn superoxide dismutase (SOD) were reduced in DL. However, six germin-like proteins (GLPs), four Cu/ZnSOD isoforms, three polyphenol oxidases, and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins), were desiccation-inducible. Desiccation provoked cell wall remodeling related to GLP-derived H2O2/HO● activity and pectin demethylesterification. This comprehensive study contributes to understanding the role and regulation of the main metabolic pathways during desiccation aiming at crop drought tolerance improvement.

Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions. However, the appearance of the DEPMPO/OOH adduct could also be observed, due to the production of O(2)(-). when endogenous SOD has been inactivated. Also, O(2)(-). was converted to .OH in an in vitro horseradish peroxidase (HRP)/H(2)O(2) system to which exogenous SOD has been added. Taken together with the discovery of the cell wall-bound Mn-SOD isoform, these results support the role of such a cell wall-bound SOD in the formation of .OH jointly with the cell wall-bound POD. According to the above findings, it seems that the hydroxycinnamic acids from the cell wall, acting as reductants, contribute to the formation of H(2)O(2) in the presence of O(2) in an autocatalytic manner, and that POD and Mn-SOD coupled together generate .OH from such H(2)O(2).

Antioxidative response in variegated Pelargonium zonale leaves and generation of extracellular H2O2 in (peri)vascular tissue induced by sunlight and paraquat.

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (>1100µmolm-2s-1 of photosynthetically active radiation) with and without paraquat (Pq), with the aim to elucidate the mechanisms of H2O2 regulation in green and white tissues with respect to the photosynthetically-dependent generation of reactive oxygen species (ROS). Sunlight induced marked accumulation of H2O2 in the apoplast of vascular and (peri)vascular tissues only in green sectors. This effect was enhanced by the addition of Pq. In the presence of diphenyl iodide, an NADPH oxidase inhibitor, H2O2 accumulation was abolished. Distinct light-induced responses were observed: in photosynthetic cells, sunlight rapidly provoked ascorbate (Asc) biosynthesis and an increase of glutathione reductase (GR) and catalase activities, while in non-photosynthetic cells, early up-regulation of soluble ascorbate peroxidase, dehydroascorbate reductase (DHAR) and GR activities was observed. Paraquat addition stimulated DHAR and GR activities in green sectors, while in white sectors activities of monodehydroascorbate reductase, DHAR and class III peroxidases, as well as Asc content rapidly increased. Differential antioxidative responses in the two tissues in the frame of their contrasting metabolisms, and the possible role of (peri)vascular H2O2 in signaling were discussed.

Carbon allocation from source to sink leaf tissue in relation to flavonoid biosynthesis in variegated Pelargonium zonale under UV-B radiation and high PAR intensity.

We studied the specific effects of high photosynthetically active radiation (PAR, 400-700 nm) and ecologically relevant UV-B radiation (0.90 W m(-2)) on antioxidative and phenolic metabolism by exploiting the green-white leaf variegation of Pelargonium zonale plants. This is a suitable model system for examining "source-sink" interactions within the same leaf. High PAR intensity (1350 µmol m(-2) s(-1)) and UV-B radiation induced different responses in green and white leaf sectors. High PAR intensity had a greater influence on green tissue, triggering the accumulation of phenylpropanoids and flavonoids with strong antioxidative function. Induced phenolics, together with ascorbate, ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6) provided efficient defense against potential oxidative pressure. UV-B-induced up-regulation of non-phenolic H2O2 scavengers in green leaf sectors was greater than high PAR-induced changes, indicating a UV-B role in antioxidative defense under light excess; on the contrary, minimal effects were observed in white tissue. However, UV-B radiation had greater influence on phenolics in white leaf sections compared to green ones, inducing accumulation of phenolic glycosides whose function was UV-B screening rather than antioxidative. By stimulation of starch and sucrose breakdown and carbon allocation in the form of soluble sugars from "source" (green) tissue to "sink" (white) tissue, UV-B radiation compensated the absence of photosynthetic activity and phenylpropanoid and flavonoid biosynthesis in white sectors.