Arabidopsis thaliana (ecotype Columbia) was grown in soil culture with 10 h light/14 h darkness at 22/18°C, respectively, and a photosynthetic photon fluence rate of 120 μmol quanta m^-2 s^-1. 6 week old plants were used for the chloroplast isolation. Leaves were harvested and homogenized in buffer containing 0.3 M sorbitol, 20 mM Tricine/KOH (pH 8.4), 5 mM ethylenediaminetetraacetic acid (EDTA) and 2 mM ascorbic acid. The homogenate was filtered through eight layers of muslin cloth and nylon mesh. The debris was removed by centrifugation at 3000 rpm and 4°C for 2 min. The sedimented chloroplasts were resuspended in isolation buffer, containing 0.33 M sorbitol, 5 mM MgCl₂, 20 mM HEPES/KOH (pH 7.9), 2 mM EDTA with freshly added ascorbate. The resuspended chloroplasts were loaded on top of a Percoll step gradient consisting of layers with 40 and 80% Percoll medium containing 0.02 g Ficoll and 0.1 g PEG. The gradient was centrifuged at 3000 rpm for 30 min without brakes. Intact chloroplasts were collected from the interphase between the Percoll layers and washed twice by spinning at 3000 rpm for 2 min. The stroma proteins were extracted following lysis and RubisCO was partially removed according to Ströher and Dietz (2008). The purity of stromal protein preparation was verified by using organelle specific enzymatic and antibody assay (Figure 1). The cytosolic marker enzyme UDP-glucose pyrophosphorylase (UGPase) activity was measured according to Zrenner et al. (1993). Using UDP-glucose and pyrophosphate as substrates, Glc-1-P released by UGPase was converted to glucose-6-phosphate (Glc-6-P) which was quantified by coupling to NADP⁺ reduction by Glc-6-P dehydrogenase. Mitochondrial type II peroxiredoxin F (AtPrxIIF) was used as a marker for mitochondrial contaminations. Equal amounts of total plant and stromal proteins (25 μg) were loaded and separated on reducing SDS-PAGE gels. Western blot analysis with antibodies raised against heterologous expressed AtPrxII F was performed as described in Finkemeier et al. (2005).

Total sulfhydryl contents of stroma proteins were determined as described by Tietze (1969). Proteins were precipitated in 3% trichloroacetic acid (TCA) and recovered after a brief centrifugation. To expose buried thiol groups the resulting pellet was dissolved in denaturing buffer containing 100 mM Tris-HCl (pH 8.0), 6 M guanidinium HCl or 1% SDS. The free thiol groups were quantified spectrophotometrically at 412 nm using 6 mM 5,5-dithio-bis(2-nitrobenzoic acid; DTNB) as substrate.

Partial oxidation in vitro was performed in 20 mM Tris-HCl (pH 7.8) buffer by adding H₂O₂ in varying stoichiometric quantities equivalent to 1, 2.5, 5, and 10% of the protein thiol content determined by DTNB assay in the respective fraction. The range of ratios which was usually equivalent to 1–10 μM concentrations was selected to identify preferred sites for oxidation. For in vivo oxidation, 50 μM MV supplemented with 0.1% Tween-20 was sprayed on whole plants that were harvested after 10, 30 and 60 min. Three independent experiments were performed for both in vitro and in vivo oxidation treatments and the identified target proteins are representative of respective replicate experiments.

All buffers were depleted from dissolved O₂ by bubbling with argon gas at room temperature (RT). The stroma protein fraction was completely reduced in the presence of 25 mM dithiothreitol (DTT). The reaction was performed inside a closed microaerobiosis chamber continuously flushed with nitrogen gas produced in a nitrogen generator to maintain the oxygen content at less than 0.4%. Excess DTT was removed by desalting using 10 ml desalting columns. The proteins in 20 mM Tris-HCl (pH 7.8) buffer with about 100 μM thiols were treated with H₂O₂(about 10 μM) for 5 min with gentle shaking to identify the initial targets of oxidation. Remaining cysteinyl thiols were alkylated using 100 mM N-ethylmaleimide (NEM) in darkness for 1 h to prevent oxidation. Excess NEM was removed by TCA precipitation according to Muthuramalingam et al. (2010). The washed precipitate was solubilized in denaturing buffer containing 200 mM Bis-Tris (pH 6.5), 6 M urea, 0.5% (w/v) SDS and 10 mM EDTA supplemented with 100 mM DTT to allow full reduction of oxidized thiols. Excess DTT was removed by repeated TCA precipitation. The labeling of the rereduced, previously oxidized thiols was achieved with 25 mM biotin maleimide in the dark for 90 min under constant shaking. Excess labeling reagent was removed either by TCA precipitation or with PD-10 desalting columns (GE Healthcare) depending on the downstream processing.

To monitor the in vivo redox status of proteins, proteins from MV-treated plants were extracted in the presence of 100 mM NEM. Furtheron, the proteins were reduced with DTT and subsequently labeled with biotin maleimide as described above. Biotinylated proteins were separated either by one or two-dimensional gel electrophoresis (2DE) and subsequently transferred onto nitrocellulose membrane using the semidry blotter Fastblot B44 (Whatman/Biometra, Germany). After a blocking step with 1% fish gelatin (Sigma, Germany) for 2 h at RT, the membrane was probed with anti-biotin antibody (Clone BN-34 from Sigma–Aldrich, St. Louis, USA). Then the membrane was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse antibody (Sigma–Aldrich, St. Louis, USA) and developed using the enhanced chemiluminescence method (Thermo Scientific, Germany).

The protein samples were desalted to remove labeling solution. This was needed since denaturing reagents used for solubilizing TCA-precipitated protein pellets inhibited binding of biotinylated polypeptides to the streptavidin column. Biotinylated proteins were enriched using streptavidin agarose. The biotinylated sample was incubated with streptavidin agarose equilibrated with phosphate buffered saline (PBS) buffer, pH 7.4, at 4°C with constant shaking overnight. Nonspecifically bound proteins were washed with 1× PBS until the absorbance at 280 nm reached zero. Proteins were incubated with elution buffer containing 1% SDS, 30 mM biotin (pH 12) for 15 min at RT, followed by heating at 96 °C for 15 min.

Proteins purified by streptavidin agarose were resolved by one-dimensional SDS-PAGE and stained with silver nitrate according to Blum et al. (1987). Spots of interest were excised from the gel and placed in U-shaped microtiter plate wells (Greiner Bio-one, Germany). To remove the silver, the excised gel spots were de-stained using Farmer’s reducing reagent containing 30 mM potassium ferricyanide (III) and 100 mM sodium thiosulfate. Then the gel spots were washed several times with ultrapure H₂O until the gel slices became transparent. Protein spots were washed twice with 30% (v/v) acetonitrile in 0.1 M ammonium hydrogen carbonate and subsequently dried in the speedvac. The gel pieces were rehydrated in the presence of 0.01 μg trypsin/μl (Promega, Mannheim, Germany) at RT for 30 min followed by overnight incubation at 37°C according to manufacturer’s protocol. The gel slices were vacuum dried, and the peptides were extracted with 50% acetonitrile and 0.1% trifluoroacetic acid for MS analysis. Acquisition of peptide mass fingerprint data and corresponding LIFT spectra was performed using an ultrafleXtreme matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) device (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam-II laser with a repetition rate of 1000 Hz. The spectra were calibrated using external calibration and subsequent internal mass correction. For databank searching, Biotools 3.2 software (Bruker Daltonics) with the implemented MASCOT search engine (Matrix Science) was used, searching for A. thaliana in the non-redundant National Center for Biotechnology Information database (26/07/2010, 55602 sequences). Search parameters were as follows: monoisotopic mass accuracy; 50 ppm tolerance; fragment tolerance of 0.3 Da; missed cleavages 1; and the allowed variable modifications were oxidation (Met), propionamide (Cys), and carbamidomethyl (Cys). Proteins were identified from all three independent experiments applying MASCOT significance scores of 60 (protein level) and 32 (peptide level). Proteins found in just one of the experiments are indicated in table legends (Tables 1–3).

The extent of biotinylation was quantified using the HABA-avidin assay developed by Green (1975). HABA forms a red complex with avidin that can be monitored spectrophotometrically at 500 nm. Due to its higher affinity, biotin displaces HABA, accompanied by the decrease in absorbance at 500 nm. The biotinylated protein samples were desalted to remove the excess of biotin maleimide reagent before performing the assay. The assay mixture consisted of 1× PBS buffer containing HABA-avidin reagent (Sigma, Germany) and 10 μg of biotinylated protein sample. After 2 min incubation the absorbance at 500 nm was recorded. The change in absorbance at 500 nm is proportional to the amount of biotin in the assay. A standard curve was generated using free biotin and used to estimate the number of moles of biotin incorporated after biotinylating the protein.

Chloroplasts were isolated and lysed to obtain a stromal protein fraction which was checked for contaminations by other cellular constituents. Type II peroxiredoxin F (AtPrxII F) was used as a marker for mitochondrial contamination using Western blot analysis. In total plant protein extract AtPrxII F was detected at the expected size of about 21 kDa, while it was absent in the stromal fraction (Figure 1A). As shown in Figure 1B the plant protein extract exhibited high rates of nicotinamide adenine dinucleotide phosphate (NADPH) formation at 340 nm, representative for high UGPase activity, while its enzymatic activity in the stromal fraction was minimal with less than 6% relative contamination.

The present study aimed to identify the primary protein targets of H₂O₂ oxidation in the chloroplast in vitro and in vivo. Percoll-purified intact chloroplasts were lysed, and the stroma protein fraction was recovered by centrifugation. Total thiol contents of stroma protein extract was determined using the DTNB assay in order to adjust the amount of H₂O₂ to be added for oxidation to 10% of total protein thiols. Protein thiols were quantified under reducing and denaturing conditions to obtain an average amount of thiols to be used as a conversion factor for future experiments. Low molecular weight thiol metabolites such as glutathione were removed by TCA precipitation, followed by a centrifugation. Both denaturing methods, namely guanidinium hydrochloride- and SDS-treatment gave a highly similar result of 57.1 ± 4.3 and 58.3 ± 3.1 μmol/g protein, respectively (mean ± SD of n = 3), corresponding to an average of 3 Cys per 50 kDa protein.

In order to optimize the workflow (Figure 2A) and to check for reaction specificity, 2-cysteine peroxiredoxin (2-Cys Prx) was used as test protein, since it is a well characterized redox-regulated protein (Figure 2B). 2-Cys Prx has two cysteinyl residues and forms an intermolecular disulfide bond upon oxidation. Thus the oxidized form runs as a dimer on non-reducing SDS-PAGE. To check the specificity of the labeling strategy, proteins were directly labeled with biotin maleimide after each step of the work flow and detected immunologically with antibody against biotin. As shown in Figure 2B, free thiols were not available for biotin maleimide labeling after oxidation of 2-Cys Prx as indicated by the absence of signal in the Western blot (lane 2). A similar result was observed after blocking of free thiols with NEM, an alkylating agent to prevent thiol-disulfide exchange reactions (lane 3). After reduction, Cys were efficiently labeled with biotin maleimide and strong bands were detected in the blot (lane 1 and 4). The band detected around 48 kDa corresponds to the half-oxidized dimer, since each dimer contains two catalytic sites each of which can form a disulfide bridge.

Stroma proteins were subjected to H₂O₂ oxidation, subsequently reduced and labeled with biotin maleimide. Oxidation was performed by adding H₂O₂ at amounts of varying stoichiometry relative to protein thiol contents (1, 2.5, 5, and 10%) which corresponded to 1 to 10 μM concentration. Under optimal conditions for photosynthesis, H₂O₂ concentrations are considered to be below 1 μM. H₂O₂ accumulates under stress. 10 μM H₂O₂ inhibits the Calvin cycle in isolated chloroplast by half (Kaiser, 1976; Asada, 1999; Polle, 2001). Remaining free thiols were blocked with NEM followed by reduction of reversibly oxidized proteins with DTT. The newly recovered thiol groups were then labeled with biotin maleimide. Hence biotinylated proteins corresponded to Cys-containing proteins that had been reversibly oxidized by the added H₂O₂. The increase in biotin label corresponding to increased oxidation is shown in Figure 3A. In the control reaction, the proteins were reduced and directly blocked with NEM without exposure to H₂O₂. Complete reduction and immediate blocking of free thiols in the control sample resulted in only minor incorporation of biotin maleimide into proteins (Figure 3A; lane 1). The labeling degree increased with increasing H₂O₂ concentrations.

The amount of biotin maleimide in the labeled samples was quantified with the HABA-avidin assay. Equal amounts of protein from different H₂O₂-treated and control samples were mixed with HABA-avidin reagent. The assay displayed a decrease in the absorbance that is proportional to the amount of biotin maleimide present in the sample. The degree of biotinylation was calculated as micromol biotin-labeled thiol per gram protein. As expected the increasing amounts of H₂O₂ and subsequent reduction allowed the incorporation of more biotin, representing the extent of thiol oxidation (Figure 3B).

Two-dimensional gel electrophoresis was performed in order to get insight into the complexity of the H₂O₂-mediated oxidative changes. One hundred micrograms of biotin maleimide-labeled control and H₂O₂-treated proteins were separated by 2DE and subsequently visualized by silver staining (Figure 4A). The corresponding Western blot membranes probed with anti-biotin antibody are shown in Figure 4B. The patterns of biotinylated polypeptides differed strongly between control and treated samples, while the patterns from the silver-stained gel detecting total proteins revealed a similar spot pattern despite the fact that apparently slightly more protein had been solubilized in the H₂O₂-treated sample. The direct comparison of the blots and the silver-stained gels for identifying and excising the proteins of interest appeared unreliable due to the expected background of unlabeled polypeptides. Therefore the biotinylated proteins were further enriched by purification via streptavidin agarose column chromatography.

Biotin-labeled control and H₂O₂-treated samples were purified by streptavidin agarose chromatography to separate the H₂O₂-sensitive biotinylated proteins from the complex protein mixture. Affinity-purified proteins were precipitated with TCA (10% w/v) to remove excess biotin from elution. The protein samples were resolved by one-dimensional SDS-PAGE analysis and visualized by silver staining (Figure 5A). Both control and H₂O₂-treated proteins exhibited a similar pattern on the gel, which is explained by loading equal protein amounts. However, immuno-reactive signals only appeared on the blot from H₂O₂-treated samples, confirming that the biotin labeling was linked to protein oxidation by H₂O₂ (Figure 5B). To identify proteins containing redox sensitive Cys thiols the indicated gel sections were excised from the gel. Results of MALDI-TOF/MS analysis are summarized in Table 1. In total 17 proteins were identified from affinity purified H₂O₂-treated samples. All identified proteins are located to the chloroplast. Five proteins function in the Calvin cycle (protein # 2, 5, 6, 11, and 12). The other identified proteins have various functions, such as nitrogen assimilation (proteins #1 and 14), adenosine triphosphate (ATP) synthesis (protein #15) and electron transport (protein # 17) among others. The analysed amino acid composition of these proteins revealed that except for the photosystem II (PSII) stability/assembly factor HCF136 (protein #16) one or more Cys are present in all identified proteins. To confirm oxidation-mediated Cys modification in H₂O₂-treated samples, the mass lists of unmatched peptides were compared with the predicted mass of in silico trypsin-digested and biotinylated peptides (Table 2). This approach allowed us to confirm two peptides of the large subunit (LSU) and small subunit (SSU) of RubisCO, single peptides of ferredoxin-dependent glutamate synthase (Fd-GOGAT), subunit B of GAPDH and ferredoxin-NADP oxidoreductase (FNR).

Enzymes such as phosphoribulokinase, GAPDH, and transketolase were identified as primary targets responding to reduction or oxidation of regulatory thiols. They were previously reported to be redox-regulated targets of Trx (Motohashi et al., 2001; Marchand et al., 2004). RubisCO was identified as one of the primary targets of oxidation. RubisCO plays a major role in photosynthesis, hence it is regulated by several mechanisms and redox-dependent modulation is one of them. Based on our MALDI-MS data analysis (Table 2) two Cys of RubisCO LSU C192 and C427 are predicted to be oxidized. In Chlamydomonas reinhardtii C192 was previously shown to be inactivated by arsenite (Moreno and Spreitzer, 1999). However, site-directed mutagenesis suggested that C192 lacks a role in disulphide-mediated inactivation. Rather C449 and 459 are assumed to be involved in redox-dependent catalytic inactivation and to trigger increased proteolysis of the protein (Moreno et al., 2008). In this study the identified C427 is conserved in 91% of the analyzed photosynthetic organisms (Figure A1 in Appendix). In three-dimensional structures C427, 449, and 459 are in close proximity with basic rich amino acids, which could be essential for oxidative activity of the protein. Due to the high protein abundance in the millimolar range, RubisCO can be assumed to act as redox buffer to maintain a suitable redox state of the chloroplast in the presence of transiently increased ROS release. RubisCO SSU was identified as thioredoxin target by several studies (Motohashi et al., 2001; Balmer et al., 2003). In this work C41 and 117 were found to be modified by biotin maleimide due to oxidation, although the distance between both Cys is too far from each other (11–25 Å) to form a disulfide bond (Taylor and Andersson, 1997). But these Cys might be a target for SOH oxidation or mixed-disulfide formation and function as oxidant sensor. In Chlamydomonas, Zaffagnini et al. (2012) recently reported 225 glutathionylated proteins, among them many Calvin cycle enzymes, indicating a role of glutathionylation in protecting and regulating chloroplast carbohydrate metabolism. We can exclude glutathionylation, since the proteins were desalted following the reduction with DTT (see Materials and Methods). This step also eliminated any glutathione from the extracts.

In higher plant chloroplasts, Fd-GOGAT is a major enzyme for glutamate synthesis, involved in the conversion of glutamine and 2-oxoglutarate to glutamate. Thioredoxin-mediated redox regulation of Fd-GOGAT was addressed in vitro by several studies (Lichter and Häberlein, 1998; Motohashi et al., 2001; Balmer et al., 2003). The amino acid sequence of the protein shows 24 Cys residues in the mature form, three of which were alkylated with NEM (Table 2). FNR catalyzes the electron transfer between ferredoxin and NADPH, producing reducing equivalents for chloroplast metabolism and thus represents a crucial enzyme for various pathways requiring reductants. The predicted mass of a peptide containing a Cys residue matched the measured mass value (±0.5 Da) suggesting, that this particular Cys is sensitive to oxidation (Table 2). GAPDH is subjected to post-translational modifications which involve thiol-disulfide transitions of regulatory Cys, complex formation with ribulose-5-phosphate kinase and a regulatory protein named CP12 (Scheibe et al., 2002). This mechanism allows the coordination of GAPDH redox regulation with availability of its substrate 1,3-bisphosphoglycerate.

Proof of concept is also provided by the identification of 2-Cys Prx and cylophilin 20-3. 2-Cys Prx is among the top 20 most abundant stroma proteins (Peltier et al., 2006), functions as high-affinity thiol-peroxidase (König et al., 2003), and undergoes large redox-dependent conformational changes linked to functional switches (Dietz, 2012). Here the recombinant 2-Cys Prx protein was a convincing system to test and validate the various steps of oxidation, blocking, labeling, and detection in the work flow (Figure 2B), and its recovery in vitro and in vivo shows also, that the early oxidizable proteins are indeed trapped with this method (Tables 1–3). Likewise cylophilin 20-3 is a known target of thiol/disulfide transition (Laxa et al., 2007) and this redox switch affects its peptidylprolyl-cis/trans isomerase activity and probably also its capability for protein/protein interactions.

The in vivo redox status of protein thiols was monitored using MV-mediated oxidative stress. The majority of the identified proteins are involved in antioxidant defense (Table 3). GST catalyzes hydroperoxide detoxification in the presence of glutathione. GST tau is also known to detoxify herbicides and plays a role in signal transduction (Neuefeind et al., 1997; Dixon et al., 2003). Thiol-mediated light/dark regulation of Calvin cycle enzymes is a well known process for years (Buchanan, 1980). In this line SBPase and fructose-bisphosphate aldolase were identified. SBPase is activated by disulfide reduction. The involved Cys have been identified by site-directed mutagenesis (Dunford et al., 1998). Interestingly overexpression of SBPase stimulates growth during early development and under stress suggesting that SBPase activity controls fluxes in the Calvin cycle to a major extent (Lefebvre et al., 2005; Feng et al., 2007). The sensitivity of SBPase to oxidation in vivo may provide an explanation why SBPase plays a particular role under stress with increased ROS production.

Regulators of PS II oxygen-evolving complex (OEC) such as PsbO-1, PsbP-1, and PsbQ were also identified as oxidation-sensitive proteins. A recent study has proposed the presence of an intra-molecular disulfide bond in PsbO-1 and PsbP-1 using a diagonal two-dimensional gel (Ströher and Dietz, 2008). PsbQ indeed has no Cys but could be associated and coeluted with other PS II OEC proteins. Biochemical studies showed that A. thaliana nucleoside diphosphate kinase 2 (NDPK2) is associated with MAPK-mediated H₂O₂ signaling in plants (Moon et al., 2003). Being one of the early oxidation targets, NDPK2 thiols might play a major role in the ROS-mediated signaling pathway. The in vivo experimental strategy should be improved in the future, e.g., by partial removal of RubisCO, followed by enrichment of less abundant proteins. The here applied method does not distinguish between direct oxidation by H₂O₂ and proximity-based oxidation mechanism where thiol proteins such as peroxiredoxins first react with H₂O₂ to form a SOH intermediate, which then oxidizes a thiol of another protein (König et al., 2012).