Dynamic compartment specific changes in glutathione and ascorbate levels in Arabidopsis plants exposed to different light intensities
- Elmien Heyneke†1,
- Nora Luschin-Ebengreuth†2, 3,
- Iztok Krajcer3,
- Volker Wolkinger3,
- Maria Müller3 and
- Bernd Zechmann3Email author
© Heyneke et al.; licensee BioMed Central Ltd. 2013
Received: 3 April 2013
Accepted: 16 July 2013
Published: 17 July 2013
Excess light conditions induce the generation of reactive oxygen species (ROS) directly in the chloroplasts but also cause an accumulation and production of ROS in peroxisomes, cytosol and vacuoles. Antioxidants such as ascorbate and glutathione occur in all cell compartments where they detoxify ROS. In this study compartment specific changes in antioxidant levels and related enzymes were monitored among Arabidopsis wildtype plants and ascorbate and glutathione deficient mutants (vtc2-1 and pad2-1, respectively) exposed to different light intensities (50, 150 which was considered as control condition, 300, 700 and 1,500 μmol m-2 s-1) for 4 h and 14 d.
The results revealed that wildtype plants reacted to short term exposure to excess light conditions with the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol and an increased activity of catalase in the leaves. Long term exposure led to an accumulation of ascorbate and glutathione mainly in chloroplasts. In wildtype plants an accumulation of ascorbate and hydrogen peroxide (H2O2) could be observed in vacuoles when exposed to high light conditions. The pad2-1 mutant reacted to long term excess light exposure with an accumulation of ascorbate in peroxisomes whereas the vtc2-1 mutant reacted with an accumulation of glutathione in the chloroplasts (relative to the wildtype) and nuclei during long term high light conditions indicating an important role of these antioxidants in these cell compartments for the protection of the mutants against high light stress.
The results obtained in this study demonstrate that the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol is an important reaction of plants to short term high light stress. The accumulation of ascorbate and H2O2 along the tonoplast and in vacuoles during these conditions indicates an important route for H2O2 detoxification under these conditions.
KeywordsArabidopsis Ascorbate Chloroplast Glutathione High light Reactive oxygen species
Plant metabolism strongly depends on the availability of visible light as it is the driving force for photosynthesis which converts carbon dioxide into organic compounds that are used by the plant for growth and development [1–3]. The light reaction of oxygenic photosynthesis is characterized by water oxidation and by the light driven transport of the electrons from water to NADP producing a proton gradient that facilitates the synthesis of ATP [4–7]. One key problem of this process is the constant generation of reactive oxygen species (ROS) such as singlet oxygen, superoxide hydroxyl radical and hydrogen peroxide (H2O2) [5–12]. If not detoxified these substances are capable of oxidizing membrane components and proteins and can lead to the degradation of nucleic acids, lipids, pigments, membranes, proteins, RNA, and DNA, causing mutation and eventually cell death [9–11]. The generation of ROS is naturally higher when plants are exposed to high light conditions as it overstrains the electron transport chain [7, 11, 13–16]. Thus, plants have to adapt to high light conditions by several metabolic and morphological changes such as reduction in the size of the light-harvesting complex [17–19], by an increase in the number of cells layers within leaves [20, 21], and by the movement of chloroplasts and leaves in order to avoid the light source [19, 22]. Other effective defense mechanism against high light include non-photochemical quenching which dissipates excess light as heat , and the detoxification of ROS by enzymes such as catalase, superoxide dismutase, oxidoreductases or antioxidants [8–10, 12, 22, 23]. Antioxidants are molecules which are able to detoxify ROS by reducing them into less harmful substances or by inhibiting the oxidation of other molecules . Besides carotenoids and α-tocopherol, which are lipohilic antioxidants within chloroplasts , two global antagonists against ROS are the water soluble antioxidants ascorbate and glutathione. As reducing agents they play important roles during the elimination of ROS individually or through the ascorbate glutathione cycle [10, 25–28]. In opposite to carotenoids and α-tocopherol, which antioxidative effects in plants are restricted to the chloroplasts, ascorbate and glutathione and related enzymes can be found in different concentrations in the different cell compartments [29–32]. Thus ascorbate and glutathione can protect plants from oxidative damage in other cell compartments besides chloroplasts as well. Considering that H2O2 production is just as high in peroxisomes during photosynthesis as a result of glycolate oxidation in the photorespiratory pathway [15, 33] and that ROS can leak from chloroplasts and peroxisomes in other cell compartments such as vacuoles and the cytosol [34, 35], sufficient levels of ascorbate and glutathione in the different cell compartments are important for the plant to fight ROS during high light conditions. In Arabidopsis plants highest levels of ascorbate were found in peroxisomes and the cytosol and lowest ones in vacuoles with intermediate labeling in nuclei, mitochondria and plastids  whereas glutathione contents were found to be highest in mitochondria followed by nuclei, the cytosol, peroxisomes, plastids and vacuoles [31, 36]. Changes in subcellular ascorbate and glutathione contents during abiotic and biotic stress are valuable stress indicators within plants and can determine the fate of the plant during situation of oxidative stress.
The aim of this study was to investigate subcellular changes in ascorbate and glutathione contents in Arabidopsis plants during the exposure to different low and high light conditions (from 50 to 1,500 μmol m-2 s-1) in order to clarify the dynamic compartment specific protection of these key antioxidants against ROS produced during high light conditions. Plants grown at 150 μmol m-2 s-1 were considered as controls (we will refer to this light intensity as control conditions throughout the text) as this light intensity is considered to be ideal for growing Arabidopsis plants and commonly used in experiments involving Arabidopsis thaliana. Different light intensities were applied for 4 h or 14 d in order to evaluate differences in short and long term adaptation strategies of the antioxidant system to high light intensities. Additionally, we compared dynamic changes of the antioxidants ascorbate and glutathione in ascorbate and glutathione deficient mutants, vtc2-1 (60% less ascorbate than the wildtype)  and pad2-1 (80% less glutathione than the wildtype) [36, 37], respectively, in order to clarify possible compensatory effects of low ascorbate and glutathione contents by glutathione and ascorbate, respectively. Other parameters such as catalase activity and H2O2 contents, number of chloroplasts and changes in the fine structure of chloroplast were additionally monitored in order to correlate different defense and adaptation strategies of Arabidopsis plants to changes in the antioxidative protection during high and low light conditions.
Subcellular changes of ascorbate and glutathione labeling were investigated by transmission electron microscopy (TEM) 4 h and 14 d after the application of different light intensities on Arabidopsis thaliana Col-0 plants and the vtc2-1 and pad2-1 mutant. The distribution of glutathione and ascorbate specific gold labeling in wildtype plants and the mutants grown at this light intensity was similar as observed in previous studies [31, 32, 40].
Exposure to different light intensities for 4 h
Exposure to different light intensities for 14 d
Exposure to different light intensities for 4 h
Exposure to different light intensities for 14 d
Chloroplast number and fine structure
Catalase activity and H2O2content
After the exposure of wildtype plants to high light for 14 d most cell compartments contained highest subcellular glutathione and ascorbate contents when plants were exposed to a light intensity of 300 μmol m-2 s- or 700 μmol m-2 s-1 and a strong decrease after the exposure to a light intensity of 1,500 μmol m-2 s-1. These changes correlated with a significant reduction of Chl and carotenoid contents and the accumulation of anthocyanins after the exposure of wildtype plants to a light intensity of 700 and 1,500 μmol m-2 s-1 for 14 d. These results complement previous studies that have demonstrated increased global ascorbate levels in whole leaves exposed to long term high light conditions [38, 39, 51–53] and correlate well with the increase of the overall ascorbate labeling of up to 200% calculated for the whole leaf in this study. Excess light represents a potential danger to the plant as it leads to the accumulation of ROS by overstraining the reactions in chloroplasts during photosynthesis [23, 43, 44]. Thus, an accumulation of ascorbate and glutathione, especially in chloroplasts as observed in wildtype plants that were exposed to a light intensity of 1,500 μmol m-2 s-1 seems to be a logical consequence in order to avoid an excess production of ROS. As H2O2 contents remained at control levels in wildtype plants exposed to excess light for 14 d it seems that the observed compartment specific adaptation in ascorbate and glutathione contents as well as modification of leaf structure (increase in leaf thickness), accumulation of anthocyanins in vacuoles, decrease in Chl contents, and adaptations of chloroplast fine structure were sufficient for the adaptation of wildtype plants to excess light conditions to avoid excess production of H2O2. Chloroplasts of wildtype plants showed a strong decrease in Chl and carotenoid contents with increasing light conditions which was similar as observed under comparable conditions  and correlated with a decrease in thylakoid contents, chloroplast size and starch contents in the wildtype. Similar effects have been observed during drought stress in spinach and spruce tree where a decrease in starch contents, chloroplast size and an increase in plastoglobuli size could be observed [54, 55]. An increase in plastoglobuli size was also observed in the present study in correlation with the exposure to high light conditions and decreasing thylakoid contents. Such a correlation seems likely during high light conditions as plastoglobuli are considered to be an important storage subcompartment of degrading thylakoid membranes  and play an important role in the breakdown of carotenoids .
In order to clarify the compartment specific importance of ascorbate and glutathione in the protection against high light conditions, subcellular ascorbate and glutathione contents were also evaluated in mutants deficient in ascorbate and glutathione contents. The glutathione deficient pad2-1 mutant, showed a significant increase in glutathione and ascorbate contents only in mitochondria (up to 147% and 42% respectively) after the exposure to different high light regimes for 4 h which highlights the importance of glutathione and ascorbate in mitochondria for cell survival in situation of stress. The pad2-1 mutant, which develops a phenotype similar to the wildtype, is characterized by glutathione levels similar to Col-0 in mitochondria, despite a strong drop of glutathione in all other cell compartments of up to 90% [36, 37]. In previous studies we have demonstrated that distorted plant development is correlated with low glutathione contents in mitochondria as the glutathione deficient rml1 mutant which develops a severe phenotype [58–60] shows a decrease in glutathione contents in all cell compartments including mitochondria of up to 97% . Thus, we can conclude that the accumulation of antioxidants in mitochondria during high light conditions in the pad2-1 mutant seems to be an important mechanism for the survival of plants with low glutathione contents during situation of stress. Long term exposure to high light conditions induced a strong decrease of glutathione and ascorbate contents in the pad2-1 mutant. Only chloroplasts and peroxisomes reacted with unchanged levels of glutathione and ascorbate, respectively. Nevertheless, in comparison to the wildtype which showed a strong decrease in ascorbate contents in peroxisomes the pad2-1 mutant showed about 470% higher levels of ascorbate in peroxisomes after the exposure to a light intensity of 1,500 μmol m-2 s-1 for 14 d. Such effects could not be found for chloroplasts, where ascorbate contents were about 50% lower than in the wildtype which indicates that the accumulation of ascorbate in peroxisomes in the glutathione deficient pad2-1 mutant might be an important long term strategy to detoxify H2O2 which is produced in peroxisomes under high light conditions [45–47]. This conclusion is supported by the observation that chloroplast number, fine structure, Chl contents, H2O2, catalase behaved similar as the wildtype and the vtc2-1 mutant to high light conditions, indicating that the pad2-1 did not suffer more oxidative stress than the other plants. Nevertheless, the pad2-1 mutant had thicker leaves with two palisade cell layers than the wildtype and the vtc2-1 mutant which both had only one palisade cell layer when exposed to a light intensity of 1,500 μmol m-2 s-1 for 14 d. Thus the increase in leaf thickness at this light intensity seems to be an additional strategy of the pad2-1 mutant in order to adapt to high light conditions. An increase in leaf thickness and higher leaf mass per unit area due to more cell layers is a commonly observed adaptation strategy to high light conditions also in other plant species [20, 21].
The ascorbate deficient vtc2-1 mutant reacted to high light conditions with a surprising strong increase of ascorbate in most cell compartments during long and short time exposure, despite alterations in ascorbate synthesis . Glutathione was increased in most cell compartments after the exposure to high light conditions for 14 d but remained mostly unchanged during short term exposure. These results correlated well with the calculated overall labeling density of ascorbate and glutathione, which was significantly increased of up to 50% and 215%, respectively, when vtc2-1 mutants were exposed to high light conditions for 14 d. These results are in line with an accumulation of total ascorbate and glutathione contents in vtc2-2 mutants observed after the long term exposure to high light [38, 39] but extend the data on the subcellular level. There it becomes obvious that the strongest accumulation of glutathione in the vtc2-1 mutant was found in chloroplasts where an increase of 600% could be observed when plants were exposed to a light intensity of 1,500 μmol m-2 s-1 for 14 d. Additionally, ascorbate accumulated in chloroplasts under these conditions indicating that the accumulation of both ascorbate and glutathione in chloroplasts are an important adaptation strategy of the vtc2-1 mutant to high light stress. The vtc2-1 mutants contain about 60–80% less ascorbate under low light conditions than the wildtype [32, 38] and despite an increase in ascorbate levels during high light stress, vtc2-1 plants did not reach wildtype levels. Thus, the accumulation of glutathione in chloroplasts and also in the other cell compartments observed in this study seem to be an important adaptation strategy of the vtc2-1 mutant especially as other mechanisms could be ruled out in this study as catalase, Chl contents, thylakoid contents, leaf anatomy, chloroplast number and the accumulation of H2O2 behaved similar to what has been observed in the wildtype and the pad2-1 mutant. Another interesting aspect in the vtc2-1 mutant is the general accumulation of glutathione (and ascorbate) in nuclei at high light conditions reaching or even succeeding levels found in the wildtype. It has been proposed previously that the accumulation of reduced glutathione could serve to protect DNA and redox-sensitive nuclear proteins from oxidation, as well as driving glutaredoxin-related processes. This will influence the binding of transcription factors which will results in adaptations of gene expression patterns. Additionally, glutathione can bind to nuclear proteins and protect them from oxidation [25, 62–66]. An accumulation of glutathione in nuclei, followed by a depletion from the cytosol has also been related to increased synthesis and rapid accumulation of cellular glutathione contents [65, 66]. Thus, it is very well likely that the massive accumulation of glutathione in nuclei of the vtc2-1 is additionally used to activate glutathione synthesis in order to increase cellular glutathione contents in this mutant. This hypothesis is supported by the general accumulation of glutathione in the vtc2-1 as discussed above and by the observation that a similar accumulation has not been observed in Col-0 and pad2-1 mutant during high light conditions.
Another interesting aspect of the adaptation of plants to high light stress was the obvious accumulation of ascorbate and glutathione inside the thylakoid lumen in wildtype, pad2-1 and vtc2-1 mutants exposed to high light conditions (Figure 3). Ascorbate inside the thylakoid lumen is important in respect to non-photochemical quenching which decreases the formation of ROS by dissipation of excess absorbed light as heat. One important mechanism for non-photochemical quenching is the formation of zeaxanthin to violaxanthin that is catalyzed by the enzyme violaxanthin de-epoxidase. This enzyme is located inside the thylakoid lumen and uses ascorbic acid as a reductant [67–70]. Additionally, ascorbate can be used as an alternative electron donor by photosystem II and I which is especially important in situation of stress when the linear electron transport is impaired [71, 72]. Thus, the accumulation of ascorbate in the thylakoid lumen of plants exposed to high light conditions and the general increase inside the stroma highlights the importance of high ascorbate contents for the compartment specific protection of chloroplasts during high light conditions.
Summing up, we can conclude that in Arabidopsis wildtype plants the accumulation of ascorbate and glutathione especially in chloroplasts, peroxisomes and the cytosol and an increased activity of catalase and enzymes involved in the ascorbate glutathione cycle are important mechanisms to protect plants against ROS produced during high light stress in the short term. Long term exposure to excess light caused an accumulation of ascorbate and glutathione in chloroplasts and several other adaptations such as the accumulation of anthocyanins in vacuoles, decrease in Chl contents and adaptations of chloroplast fine structure. Additionally, the accumulation of ascorbate in vacuoles of wildtype plants indicates an important role of this antioxidant in vacuoles for the detoxification of H2O2 leaking from peroxisomes and chloroplasts into the cytosol and vacuoles (Figure 11). The accumulation of ascorbate in peroxisomes in the glutathione deficient pad2-1 mutant and of glutathione in the chloroplasts of the ascorbate deficient vtc2-1 mutant relative to the wildtype Col-0 during long term exposure to high light conditions indicates an important role of these antioxidants in these cell compartments in order to protect the mutants against high light conditions.
After stratification for 4 d at 4°C seeds of Arabidopsis thaliana [L.] Heynh. ecotype Columbia (Col-0), the glutathione and ascorbate deficient mutants pad2-1 and vtc2-1, respectively, were grown on “Naturahum” potting soil (Ostendorf Gärtnereierden GmbH., Vechta, Germany) in growth chambers with 8/16 h day/night photoperiod. Day and night temperatures were set at 22°C and 18°C, respectively, the relative humidity was 60% and the plants were kept at 100% relative soil water content. Light intensity varied between 120 and 140 μmol m-2 s-1 (lower and upper leaves, respectively). Eight and six week old plants were exposed to different light intensities for 4 h and 14 d, respectively, in the same growth chamber: 1) 50 μmol m-2 s-1, 2) 150 μmol m-2 s-1, 3) 300 μmol m-2 s-1, 4) 700 μmol m-2 s-1, and 5) 1,500 μmol m-2 s-1. Different light intensities were applied by placing the plants at different distances below compact fluorescent lamps (Plug and Grow, 6400 K, white/blue spectrum; Agriculture Trading AG, Walenstadt, Switzerland). Temperature measured at the leaves was about 22°C in plants exposed to 50, 150, 300 μmol m-2 s-1, and about 24°C at plants exposed to 700 and 1,500 μmol m-2 s-1, respectively. Harvesting of the plants was performed about 4 h after the onset of the different light intensities. At the time of harvesting all plants were about 8 weeks old and all leaves were harvested from the 3th or 4th rosette. Care was taken that the leaves were about the same size, showed a similar developmental stage and that the areas chosen for further investigation were not shaded by other leaves.
Sample preparation for TEM and immunogold labeling
Preparation of samples for TEM, immunogold labeling of glutathione and ascorbate, and visualization of H2O2 by CeCl3 was performed as described previously [31, 32, 36, 73]. Small samples of the youngest fully developed leaves close to the middle vein (about 1.5 mm2) from at least 3 different plants were cut on a modeling wax plate either in a drop of i) 2.5% glutaraldehyde in 0.06 M Sørensen phosphate buffer at pH 7.2 for ultrastructural investigations or ii) 2.5% paraformaldehyde, 0.5% glutaraldehyde in 0.06 M Sørensen phosphate buffer at pH 7.2 for cytohistochemical investigations or iii) 5 mM cerium chloride (CeCl3) in 50 mM MOPS-buffer (pH7.2) for subcellular H2O2 visualization. Samples for ultrastructural and cytohistochemical investigations were then transferred into glass vials and fixed for 90 min at room temperature (RT) in the above mentioned solutions. Samples for the visualization of subcellular H2O2 distribution were incubated with CeCl3 solution for 60 min and then fixed in 2.5% glutaraldehyde in 0.06 M Sørensen phosphate buffer at pH 7.2 for 90 min at RT. For ultrastructural analysis and H2O2 localization samples were then rinsed in buffer (4 times for 15 min each) and post-fixed in 1% osmium tetroxide in 0.06 M Sørensen phosphate buffer for 90 min at RT. The samples were then dehydrated in a graded series of increasing concentrations of acetone (50%, 70%, 90%, and 100%). Pure acetone was then exchanged for propylene oxide and the specimens were gradually infiltrated with increasing concentrations of Agar 100 epoxy resin (30%, 60%, and 100%) mixed with propylene oxide for a minimum of 3 h per step. Samples were finally embedded in pure, fresh Agar 100 epoxy resin (Agar Scientific Ltd, Stansted, UK) and polymerized at 60°C for 48 h. For cytohistochemical investigations samples were rinsed in 0.06 M Sørensen phosphate buffer (pH 7.2) for 4 times 15 min after fixation. They were then dehydrated in increasing concentrations of acetone (50%, 70%, and 90%) at RT for 20 min at each step. Subsequently, specimens were gradually infiltrated with increasing concentrations of LR-White resin (30%, 60% and 100%; London Resin Company Ltd., Berkshire, UK) mixed with acetone (90%) for a minimum of 3 h per step. Samples were finally embedded in pure, fresh LR-White resin and polymerized at 50°C for 48 h in small plastic containers under anaerobic conditions. Ultrathin sections (80 nm) were cut with a Reichert Ultracut S ultramicrotome (Leica Microsystems, Vienna, Austria).
Determination of chloroplast number and their fine structures
Changes in the number of chloroplasts and their inner structures were evaluated according to Zechmann et al.  by investigating four different leaf samples from wildtype plants and mutants grown in different light intensities for 14 d. Chloroplast number and inner structures were not further evaluated in plants exposed to different light intensities for 4 h as no obvious differences could be found. An Olympus AX70 light microscope (Olympus, Life and Material Science Europa GmbH, Hamburg, Germany) with a 40× objective lens (n.a. 0.5-1.35) was used to determine the number of sectioned chloroplasts in the palisade cell layer and the spongy parenchyma by counting the chloroplasts per cell on 4 semithin cross-sections (3 μm) for each replicate sample. A minimum of 100 cells per leaf-type were examined to calculate the number of sectioned chloroplasts in the cells. Ultrathin sections were investigated with the TEM to determine changes in the ultrastructure of the chloroplasts including the thylakoid-system, starch grains, and plastoglobuli. These structures were then analyzed as digital images using the program Optimas 6.5.1 (BioScan Corp.). A minimum of 20 sectioned chloroplasts from at least 10 different cells from four different samples per leaf-type were examined.
Immunogold labeling of glutathione and ascorbate
Immunogold labeling of glutathione and ascorbate was done according to Zechmann et al. [32, 36] with ultrathin sections on coated nickel grids with the automated immunogold labeling system Leica EM IGL (Leica, Microsystems, Vienna, Austria). The ideal dilutions and incubation times of the primary (anti-ascorbate IgG; Abcam plc, Cambridge, UK; anti-glutathione rabbit polyclonal IgG, Millipore Corp., Billerica, MA, USA) and secondary antibodies (goat anti rat IgG and goat anti rabbit both from British BioCell International, Cardiff, UK) were determined in preliminary studies by evaluating the labeling density after a series of labeling experiments. The final dilution of primary and secondary antibodies used in this study showed a minimum of background labeling outside the sample with a maximum of specific labeling in the sample. The sections were blocked for 20 min with 2% bovine serum albumine (BSA, Sigma-Aldrich, St. Louis, MO, USA) in phosphate buffered saline (PBS, pH 7.2) and then treated with the primary antibodies against ascorbate diluted 1:300 in PBS containing 1% BSA and glutathione diluted 1:50 in PBS containing 1% goat serum for 2 h at RT. After a short rinse in PBS (3 times 5 min each), samples were incubated with a 10 nm gold-conjugated secondary antibodies (goat and rat IgG for ascorbate labeling and goat anti rabbit IgG for glutathione labeling) diluted 1:50 (for sections incubated with the glutathione antibody) and 1:100 (for sections incubated with the ascorbate antibody) in PBS for 90 min at RT. After a short wash in PBS (3 times 5 min), and distilled water (2 times 5 min) labeled grids were either immediately observed in a Philips CM10 transmission electron microscope or post stained with uranyl-acetate (2% dissolved in aqua bidest) for 15 s.
Micrographs of randomly photographed immunogold labeled sections in palisade parenchyma cells were digitized and gold particles were counted automatically using the software package Cell D with the particle analysis tool (Olympus, Life and Material Science Europa GmbH, Hamburg, Germany) in different visually identified and manually traced cell structures (mitochondria, plastids, nuclei, peroxisomes, the cytosol, vacuoles). Unspecific background labeling was determined on the sections (outside the specimen) and subtracted from the values obtained in the sample. A minimum of 20 (peroxisomes and vacuoles) to 60 (other cell structures) sectioned cell structures of at least 15 different cells were analyzed for gold particle density per sample. The obtained data were statistically evaluated using Statistica (Stat-Soft Europe, Hamburg, Germany).
Several negative controls were made to support the specificity of the immunogold procedure. Negative controls were treated either with (i) gold conjugated secondary antibody (goat anti rat IgG for ascorbate and goat anti rabbit IgG for glutathione) without prior incubation of the section with the primary antibodies, (ii) non specific secondary antibody (goat anti rabbit IgG for ascorbate and goat anti rat IgG for glutathione), (iii) preimmune serum instead of the primary antibody and (iv) primary antibody against ascorbate and glutathione pre-adsorbed with an excess of reduced and oxidized ascorbate and glutathione, respectively for 2 h prior to labeling of the sections. For the latter a solution containing either 10 mM of ascorbic acid, dehydroascorbic acid, reduced or oxidized glutathione was incubated with or without 0.5% glutaraldehyde for 1 h. When glutaraldehyde was used then its excess was saturated by incubation for 30 min in a solution of 1% (w/v) BSA. The resulting solutions were both used in independent experiments to saturate the anti-ascorbate and glutathione antibodies for 2 h prior to its use in the immunogold labeling procedure described above. Labeling on sections treated as negative controls showed no or only very little gold particles bound to ascorbate and glutathione which was similar to previous results obtained by using the same methods in different plant species [31, 32]. The specificity and accuracy of the immunogold localization method for ascorbate and glutathione used in this study has been demonstrated in detail in previous works [31, 32, 36]. The immunogold localization of ascorbate in mutants deficient in ascorbate (vtc2-1 and vtc2-2) revealed a strong decrease of subcelluar ascorbate specific labeling between 50 to 60% when compared to Arabidopsis thaliana Col-0 plants. This data correlated well with biochemical measurements which revealed a similar decrease of ascorbate contents in whole leaves of these mutants . The specificity and accuracy of the immunogold labeling method for glutathione was demonstrated on glutathione deficient mutants pad2-1 and rml1 which both showed a strong decrease of compartment specific glutathione labeling of up to 91% and 98%, respectively. This data correlated well with biochemical measurements of glutathione in these mutants revealing a similar decrease in whole leaves of these mutants [37, 59, 60]. Further studies using rapid fixation methods (e.g. high pressure freezing, microwave assisted fixation) revealed that ascorbate and glutathione were not redistributed or washed out during chemical fixation at RT which takes 90 min as labeling density and the ratio of labeling between cell compartments remained the same when these methods were used [31, 32].
Determination of Chl a/b and carotenoids
Plant tissues were frozen and ground in liquid nitrogen. Chl and carotenoids were extracted with 100% acetone in darkness at 4°C for 20 min. The homogenate was centrifuged and pigment content was quantified spectrophotometrically by measuring the absorbance at 663, 645 and 470 nm on a UV-spectrophotometer (Hitachi U-3000). Pigment content was calculated as described previously .
Activity of catalase
Catalase activity was measured as previously described . Leaf material was frozen in liquid nitrogen, freeze dried and ground with an oscillating mill (Retsch MM400). 75 mg insoluble polyvinylpyrrolidone (PVP) was added to 20 mg plant material and the powder was extracted into 2.2 ml 0.1 M NaH2PO4 (pH 7.5), 1 mM EDTA. Samples were centrifuged for 10 min at 14000 rpm at 4°C and the supernatant was used for further investigations. Catalase activity was measured with a photometer (Hitachi U3000) as the absorbance decrease at 240 nm (ϵ240 = 0.04 mM-1 cm-1) at 25°C in 50 mM KH2 PO4 and 40 mM H2O2 against reagent blank.
Content of H2O2
H2O2 contents were measured as previously described . Leaf material was frozen in liquid nitrogen, freeze dried and ground with an oscillating mill (Retsch MM400). 50 mg plant material was extracted in 1 ml 0.2 M perchloric acid and centrifuged at 14000 rpm for 10 min at 4°C. The supernatant was neutralized with a 4 M sodium hydroxide solution (pH 7.5) and centrifuged at 14000 rpm for 10 min at 4°C. The supernatant was used for further analysis as described previously . Briefly, 300 μl of the supernatant was mixed with 100 μl of a known amount of hydrogen peroxide, 100 μl 2% potassium iodide solution, 100 μl of 2 M hydrochloric acid. The mixture was incubated for 10 min on a shaker and then 50 μl of 0.01% toluidine blue indicator solution followed by 200 μl 2 M sodium acetate solution and 150 μl distilled water were added. Absorbance was measured with a photometer (Hitachi U3000) at 628 nm against a reagent blank.
Bovine serum albumin
Phosphate buffered saline
Reactive oxygen species
Transmission electron microscopy
This work was supported by the Austrian Science Fund (FWF, P22988 to B.Z.).
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