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Fingerprinting antioxidative activities in plants.

Saleh L, Plieth C - Plant Methods (2009)

Bottom Line: 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.TAC runs in parallel with GR.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Biochemie und Molekularbiologie, Universität Kiel, Am Botanischen Garten 9, 24118 Kiel, Germany. cplieth@zbm.uni-kiel.de.

ABSTRACT

Background: 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.

Results: 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.Experiments with Lepidium sativum 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Quenching of CTZ chemiluminescence by superoxide scavengers from Lepidium. CTZ was mixed with hypoxanthine at t = 12 s. This gave a background luminescence due to the presence of ambient oxygen. The superoxide yielding reaction was started by injection of xanthine oxidase to the assay mix at t = 48 s. Light output was quenched by an extract from Lepidium containing superoxide scavengers at t = 72 s (green line). The red line represents non-enzymatic scavenging of a heat-inactivated (30 min at 95°C) sample. The grey line is the control experiment with buffer injected. The steady state luminescence after starting the reaction with XOD (62 s < t < 72 s) was used to normalise the data.
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Figure 3: Quenching of CTZ chemiluminescence by superoxide scavengers from Lepidium. CTZ was mixed with hypoxanthine at t = 12 s. This gave a background luminescence due to the presence of ambient oxygen. The superoxide yielding reaction was started by injection of xanthine oxidase to the assay mix at t = 48 s. Light output was quenched by an extract from Lepidium containing superoxide scavengers at t = 72 s (green line). The red line represents non-enzymatic scavenging of a heat-inactivated (30 min at 95°C) sample. The grey line is the control experiment with buffer injected. The steady state luminescence after starting the reaction with XOD (62 s < t < 72 s) was used to normalise the data.

Mentions: Due to its very short lifetime, O2-· has to be generated in situ. The most convenient method to generate O2-· employs xanthine oxidase (XOD) [47]. While XOD is generating O2-· (Fig. 3.1A in additional file 3) and thus producing light in presence of CTZ (Fig. 3.1B in additional file 3), any O2-·-scavenger in the assay reduces the steady state O2-· concentration and thus light output. This quenching of CTZ luminescence is used to quantify the scavenger activity. In the dialysed sample non-enzymatic high molecular weight scavengers [48] can be distinguished from enzymatic (i.e. SOD) scavenging activities (Figure 3) (also see Fig. 3.2 in additional file 3).


Fingerprinting antioxidative activities in plants.

Saleh L, Plieth C - Plant Methods (2009)

Quenching of CTZ chemiluminescence by superoxide scavengers from Lepidium. CTZ was mixed with hypoxanthine at t = 12 s. This gave a background luminescence due to the presence of ambient oxygen. The superoxide yielding reaction was started by injection of xanthine oxidase to the assay mix at t = 48 s. Light output was quenched by an extract from Lepidium containing superoxide scavengers at t = 72 s (green line). The red line represents non-enzymatic scavenging of a heat-inactivated (30 min at 95°C) sample. The grey line is the control experiment with buffer injected. The steady state luminescence after starting the reaction with XOD (62 s < t < 72 s) was used to normalise the data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656482&req=5

Figure 3: Quenching of CTZ chemiluminescence by superoxide scavengers from Lepidium. CTZ was mixed with hypoxanthine at t = 12 s. This gave a background luminescence due to the presence of ambient oxygen. The superoxide yielding reaction was started by injection of xanthine oxidase to the assay mix at t = 48 s. Light output was quenched by an extract from Lepidium containing superoxide scavengers at t = 72 s (green line). The red line represents non-enzymatic scavenging of a heat-inactivated (30 min at 95°C) sample. The grey line is the control experiment with buffer injected. The steady state luminescence after starting the reaction with XOD (62 s < t < 72 s) was used to normalise the data.
Mentions: Due to its very short lifetime, O2-· has to be generated in situ. The most convenient method to generate O2-· employs xanthine oxidase (XOD) [47]. While XOD is generating O2-· (Fig. 3.1A in additional file 3) and thus producing light in presence of CTZ (Fig. 3.1B in additional file 3), any O2-·-scavenger in the assay reduces the steady state O2-· concentration and thus light output. This quenching of CTZ luminescence is used to quantify the scavenger activity. In the dialysed sample non-enzymatic high molecular weight scavengers [48] can be distinguished from enzymatic (i.e. SOD) scavenging activities (Figure 3) (also see Fig. 3.2 in additional file 3).

Bottom Line: 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.TAC runs in parallel with GR.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Zentrum für Biochemie und Molekularbiologie, Universität Kiel, Am Botanischen Garten 9, 24118 Kiel, Germany. cplieth@zbm.uni-kiel.de.

ABSTRACT

Background: 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.

Results: 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.Experiments with Lepidium sativum 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus