Fingerprinting antioxidative activities in plants

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.

Figure 1

The enhanced catalytic horseradish peroxidase cycle (adapted from [25]). Luminol (L) is used as substrate for the light generating process. A di-aza-quinone (AQ) is formed as intermediate. This in turn is oxidised by hydrogen peroxide (H₂O₂) to form an excited state of aminophtalate (AP*). The final step is the emission of blue (420 nm) light (h·ν) when the excited AP* returns to its ground state. Luminol can work as a substrate of the horseradish peroxidase (HRP). However, for analytical purposes an intermediate aromatic hydrogen donor (AH) is added. This enhancer serves as primary substrate for the HRP and its radical (A^·) subducts electrons from luminol (L) and thus forms the radical form L^·. AQ is formed by electron transfer between two L^·. The HRP compound I-state (CMP I) is sensitive to excess of H₂O₂ [33] and can undergo a peroxide inactivation (so-called 'suicide reaction'; grey arrows) when the concentration of H₂O₂ added to start the light emitting reaction is too high (compare Fig. 1.4 in additional file 1).

Figure 2

Quenching of HRP-generated light output by an antioxidant. A: Light generation of the HRP-catalysed luminol reaction was triggered by the addition of H₂O₂ (at time t₀ = -36 s). The reaction is quenched by the addition of an antioxidant when a constant output signal is established. Here Trolox™ ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) a water soluble derivative of tocopherol (vitamin E) was used. The antioxidant is oxidised by H₂O₂ thus inhibiting the HRP-catalysed reaction. When the antioxidant is depleted, the HRP reaction resumes and the light signal recovers. B: The time point of signal recovery t_r depends on the amount of added antioxidant and is defined here as the time where the rate of signal increase is maximum. The curves here are derived from the curves in A and represent the first derivative of the running mean (n = 10) on the raw data. Luminescence is given here in counts per second (cps) and its recovery in cps per s.

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.

Figure 4

Fingerprints of antioxidative activities in Lepidium sativum after abiotic stress. The percentage change in the five screened parameters is represented by the five radial axes (data from Figures 5F, 6F, 7F, and 8F). The red pentagon in each panel represents the line of no change. Black polygons represent the antioxidative status immediately after stress treatment. Grey polygons represent the status after stress recovery. Each point is average of 5 technical replicates run on biological material pooled from 3 independent experiments. Standard deviations are given in Figures 5F–8F.

Figure 5

Cold-induced alterations in the antioxidative system of Lepidium sativum. For each of the five screened parameter (TAC, LUPO, SOSA, CAT, GR) the status after stress treatment (12 h at 0°C) is given (black bars) and compared to the control of untreated plants (white bars). The grey bars represent the status of the particular parameter after 12 h recovery. For TAC (A) the recovery time in the luminescence signal was calibrated in terms of Trolox equivalents related to the protein content of the sample. The percentage change for each parameter is summarised (F) and significance according to Student's T-Test is marked with asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). Each column represents the average of five technical replicates run on pooled plant material from three independent growth and treatment experiments. Error bars represent StDv.

Figure 6

Heat-induced alterations in the antioxidative system of Lepidium sativum. TAC, LUPO, SOSA, CAT, GR were measured after heat treatment (6 h at 42°C; black bars) and compared to the control of untreated plants (white bars). The grey bars represent the status of the particular parameter after 6 h recovery. Data presentation as in Figure 5.

Figure 7

Salt-induced alterations in the antioxidative system of Lepidium sativum. Salt treatment (24 h at 150 mM NaCl in 0.5 × MS medium; black bars) is compared to the untreated plants (white bars). The grey bars represent the status after 24 h recovery. Data presentation as in Figure 5.

Figure 8

Drought-induced alterations in the antioxidative system of Lepidium sativum. Drought was induced by withdrawal of nutrient medium for 24 h and antioxidative parameters were measured (black bars) in comparison to untreated plants (white bars). The grey bars represent the recovery status 24 h after restoring the nutrient medium. Data presentation as in Figure 5.