Fingerprinting antioxidative activities in plants

<p>Abstract</p> <p>Background</p> <p>A plethora of concurrent cellular activities is mobilised in the adaptation of plants to adverse environmental conditions. This response can be quantified by physiological experiments or metabolic profiling. The intention of this work is to reduce the number of metabolic processes studied to a minimum of relevant parameters with a maximum yield of information. Therefore, we inspected 'summary parameters' characteristic for whole classes of antioxidative metabolites and key enzymes.</p> <p>Results</p> <p>Three bioluminescence assays are presented. A horseradish peroxidase-based total antioxidative capacity (TAC) assay is used to probe low molecular weight antioxidants. Peroxidases are quantified by their luminol converting activity (LUPO). Finally, we quantify high molecular weight superoxide anion scavenging activity (SOSA) using coelenterazine.</p> <p>Experiments with <it>Lepidium sativum </it>L. show how salt, drought, cold, and heat influence the antioxidative system represented here by TAC, LUPO, SOSA, catalase, and glutathione reductase (GR). LUPO and SOSA run anti-parallel under all investigated stress conditions suggesting shifts in antioxidative functions rather than formation of antioxidative power. TAC runs in parallel with GR. This indicates that a majority of low molecular weight antioxidants in plants is represented by glutathione.</p> <p>Conclusion</p> <p>The set of assays presented here is capable of characterising antioxidative activities in plants. It is inexpensive, quick and reproducible and delivers quantitative data. 'Summary parameters' like TAC, LUPO, and SOSA are quantitative traits which may be promising for implementation in high-throughput screening for robustness of novel mutants, transgenics, or breeds.</p

A novel fluorescent pH probe for expression in plants

BACKGROUND: The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. RESULTS: pHluorins have a higher pH-sensitivity. However, Pt-GFP has a broader pH-responsiveness, an excellent dynamic ratio range and a better acid stability. We demonstrate how Pt-GFP expressing Arabidopsis thaliana report cytosolic pH-clamp and changes of cytosolic pH in the response to anoxia and salt-stress. CONCLUSION: Pt-GFP appears to be the better choice when used for in vivo-recording of cellular pH in plants

Peroxide-Induced Liberation of Iron from Heme Switches Catalysis during Luminol Reaction and Causes Loss of Light and Heterodyning of Luminescence Kinetics

The peroxidation of luminol yields bright luminescence when the reaction is catalyzed by heme proteins. However, an excess of peroxide leads to less light and altered luminescence kinetics, an effect commonly referred to as “suicide inactivation”. The aim of this study is to present the molecular processes causing this effect. A comprehensive set of data reported here demonstrates that suicide inactivation is due to a peroxide-induced liberation of iron from its coordinating porphyrin. Liberated iron launches catalysis of the reaction at much lower efficiency. The light-yielding efficiencies of different organic and inorganic catalysts are precisely quantified and compared. It is shown that the catalysis by free iron involves superoxide. This is explained by the formation of a ferryl-oxo-iron complex. In this context, a complete reaction mechanism involving a modified Fenton–Haber–Weiss cycle is proposed for the first time. The switch from the highly efficient biogenically catalyzed luminescence to a less efficient inorganically catalyzed reaction is accompanied by a transition from “flash-type” to “glow-type” luminescence kinetics. Ethylenediaminetetraacetic acid-mediated chelation of iron is used to demonstrate this effect and to separate both kinetics. The explanation of kinetic heterodyning is underpinned by mathematical modeling. The results are able to explain the as yet unexplained phenomena discussed in the less recent literature and to settle disputes about them. It is concluded that peroxide concentrations exceeding the level tolerated by the catalyzing heme protein negatively impact performance and precision of luminol-based assays, where the light yield is used as a quantitative measure for analyte concentrations

Redox Modulators Determine Luminol Luminescence Generated by Porphyrin-Coordinated Iron and May Repress “Suicide Inactivation”

Iron porphyrin catalysts of the luminol reaction (horseradish peroxidase, hemoglobin, cytochrome c, and hemin) interact with diverse reducing compounds. Here, it is demonstrated how the chemiluminescence yield is modulated by such interactions. The compounds accepted as substrates protect the catalyst against the “suicide inactivation” caused by high peroxide concentrations. The reducing agents not accepted by the catalyst inhibit light production either by generating a futile redox cycle of the luminophore or by irreversibly inactivating the catalytic center. In the case of a futile cycle, light emission resumes as soon as the reducing agents in the reaction are consumed, whereas with an irreversible inactivation, light emission does not recover. The characteristics of luminescence enhancement and quenching depending on interfering agents are also reported here. They reveal details about the relative redox potentials of the involved compounds. It is discussed how this should be considered when the luminol reaction is used for quantitative analyses and when unpurified samples with a broad compound matrix are to be assayed