Glutathione oxidation in response to intracellular H₂O₂: Key but overlapping roles for dehydroascorbate reductases

<p>Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H₂O₂ detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function <i>gstU8, gstU24, gstF8, prxIIE</i> and <i>prxIIF</i> mutants as well as double <i>gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF</i> mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H₂O₂ metabolism.</p

Glutathione oxidation in response to intracellular H₂O₂: Key but overlapping roles for dehydroascorbate reductases

<p>Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H₂O₂ detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function <i>gstU8, gstU24, gstF8, prxIIE</i> and <i>prxIIF</i> mutants as well as double <i>gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF</i> mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H₂O₂ metabolism.</p

PARP inhibition reduces the transcriptional induction of anthocyanin pathway genes.

<p><i>Arabidopsis</i> plants (Col-0) were grown at 80–100 µE, 22°C on MS medium with (+3 MB) or without (−3 MB) the PARP inhibitor 3-Methoxy-benzamide (3 MB) and subjected to three different conditions: control, oxidative stress (0.1 µM Paraquat) or sucrose stress (150 mM Sucrose) and harvested after 14 days. RNA was extracted from seedlings pooled from all five replicates within one experiment, with three independent experiments (n = 3). The average relative expression, normalized against the housekeeping gene <i>PP2A</i>, is shown, for the biosynthetic (A–F) and the regulatory (G–I) genes.</p

Chemical PARP inhibition changes cellular redox profiles.

<p>Arabidopsis Col-0 seedlings were grown for 14 days at 80–100 µE, 22°C on MS media with (+3 MB) or without (−3 MB) the PARP inhibitor 3-Methoxy-benzamide (3 MB) and were subjected to three different treatments: control, oxidative stress (0.1 µM Paraquat) or sucrose stress (150 mM sucrose). Shown are (A) the NAD+ content, (B) the total ascorbate content, (C) the reduction level of the ascorbate, (D) the total glutathione and (E) the reduction of the total glutathione. Data are combined from three independent experiments with 2 replicates in each experiment (n = 6). Asterisks indicate significant difference (P<0.05) compared to Col-0 grown in the same condition without 3 MB.</p

PARP inhibitory effect on anthocyanin accumulation is associated genetically with PARP activity and based on transcriptional control.

<p>The <i>Col-0</i>, <i>PAP1-OX</i> and T-DNA insertion lines of the three <i>PARP</i> genes (<i>parp1</i>, <i>parp2</i>, <i>parp3</i>) were grown for 14 days at 80–100 µE, 22°C on MS medium with (+3 MB) or without (−3 MB) 0.2 mM of the PARP inhibitor 3-Methoxy-benzamide (3 MB) in the medium. A, the relative anthocyanin content of the <i>PAP1</i>-OX line is shown, combined from three independent experiments with three replicates in each experiment (n = 9). The reduction of ∼20% is not significant (P = 0.07) and is much smaller than the reductions after 14 days for oxidative (∼70%) or sucrose (∼50%) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037287#pone-0037287-g003" target="_blank">Fig. 3B</a>). B, the mutant lines were subjected to two different conditions; control or oxidative stress (0.1 µM Paraquat). The relative anthocyanin content is shown. Data are combined from 3 independent experiments with two to four replicate plates in each experiment (n = 9). Asterisks indicate significant difference (P<0.05) of the mutant lines compare to the Col-0 grown in the same condition.</p

Dose response of plants to chemical PARP inhibition.

<p><b>A</b>, <i>Arabidopsis thaliana</i> (Col-0) and PARPsig::RNAi (sigPARP) seedlings were grown for 21 days at 80–100 µE, 22°C on MS media containing different concentrations (mM) of the PARP inhibitor 3-Methoxy-benzamide (3 MB). The average individual fresh weight was determined by weighing 32 pooled seedlings from each plate, with four replicates (plates) in each experiment, repeated in two independent experiments (n = 8). Asterisks indicate significant (P<0.05) difference between Col-0 and PARPsig::RNAi seedlings grown without and those treated with PARP inhibitor. B, Arabidopsis plants were grown at 80–100 µE, 22°C on half MS medium and subjected to short- or long-term stress in the presence (+3 MB) or absence (−3 MB) of 0.2 mM of the PARP inhibitor 3 MB in the medium. For the short-term stress, 14 day old plants were transferred to either 450 µE (high-light) for two days and harvested at day 19 after three days of recovery or to 40°C (heat) for 6 h and harvested after seven days of recovery (day 21). For the long-term stress, plants were grown for 21 days either in control, 0.1 µM Paraquat (oxidative) or 75 mM NaCl (salt) stress conditions. Fresh weight was determined by weighing a pool of 32 seedlings from each plate, with four replicates (plates) in each experiment repeated in two independent experiments (n = 8). Asterisks indicate treatments which are significant different (P<0.05) compared to the Col-0 grown on 0 mM 3 MB.</p

Transcriptional changes induced by PARP inhibition displayed on MAPMAN.

<p>Arabidopsis plants were grown for 18 days at 80–100 µE, 22°C on half MS medium in the presence (+3 MB) or absence (−3 MB) of 0.2 mM of the PARP inhibitor 3-Methoxy-benzamide (3 MB) within the media. For the expression analysis four samples from four different plates were pooled for both conditions in each experiment and frozen immediately in liquid nitrogen, repeated in two independent experiments. The RNA was extracted according to the requirements of the Affymetrix-microarray facility at the VIB (MAF) and processed according to the manufactures instructions.</p

Metabolite changes induced by PARP inhibition and detected by GC-MS.

<p>For the GC-MS metabolite profiling samples, a total of ∼10 seedlings from 4 plates were harvested individually for both conditions (+/−3 MB) in each experiment, repeated in three individual experiments (n = 12). The plants were grown in parallel to those for the microarray expression analysis. The metabolite extraction and further processing was done at the “Plateforme Métabolisme-Métabolome (IFR87)” accordingly to (55). Metabolites that were significantly different (<i>t</i>-test, p<0.05, Bonferroni-Holm multiple testing correction) between the 3 MB treated plants (+3 MB) and the control plants (−3 MB) are shown. Values presented are the ratio (fold-change) in the abundance of +3 MB relative to −3 MB.</p

Chemical PARP inhibition alters photosynthesis.

<p><i>Arabidopsis</i> seedlings (Col-0) were grown at 80–100 µE, 22°C on MS medium with (+3 MB) or without (−3 MB) the PARP inhibitor 3-Methoxy-benzamide (3 MB) and subjected to different conditions: control and oxidative stress (0.1 µM Paraquat) and for the chlorophyll measurements also sucrose (3.5% sucrose). A, shows the non-photochemical quenching (NPQ), the proportion of open PSII reaction centers (qP) and the effective quantum yield (Y_II) measured via PAM imaging. B, shows the chlorophyll content after 8 days. For (A) 8–18 seedlings were analyzed in each of the 4 independent experiments (n = 42); for (B) data are combined from three independent experiments with 5 replicates in each experiment (n = 15). Asterisks indicate significant difference (P<0.05) between seedlings grown in the same condition but treated with 3 MB or without 3 MB.</p

PARP inhibition reduces anthocyanin accumulation under stress.

<p><i>Arabidopsis</i> plants (Col-0) were grown at 80–100 µE, 22°C on MS medium with (+3 MB) or without (−3 MB) the PARP inhibitor 3-Methoxy-benzamide (3 MB) and subjected to three different conditions: control, oxidative stress (0.1 µM Paraquat) or sucrose stress (150 mM Sucrose) and harvested after 14 days. A, indicates the whole plant phenotype of representative plants from all treatments. B, indicates the relative anthocyanin content after 14 days. Data are combined from three independent experiments with 5 replicates in each experiment (n = 15). C, shows the effect of different PARP inhibitors used: 3-Methoxy-benzamide (3 MB), 3-Methyl-benzamide (3MeB) and 3-Aminophthalaminhydrazide (3AP). The relative anthocyanin content is shown, combined from two independent experiments with 5 replicates in each experiment (n = 10). Asterisks indicate significant differences (P<0.05) of plants grown with 3 MB compared to those without 3 MB in the same condition.</p