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Keywords:

  • catalase;
  • hydrogen peroxide (H2O2);
  • nitrogen metabolism;
  • PP2A;
  • proteomics;
  • redox;
  • salicylic acid

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Oxidative stress responses are influenced by growth day length, but little is known about how this occurs. A combined reverse genetics, metabolomics and proteomics approach was used to address this question in Arabidopsis thaliana.
  • A catalase-deficient mutant (cat2), in which intracellular oxidative stress drives pathogenesis-related responses in a day length-dependent manner, was crossed with a knockdown mutant for a specific type 2A protein phosphatase subunit (pp2a-b′γ). In long days (LD), the pp2a-b′γ mutation reinforced cat2-triggered pathogenesis responses.
  • In short days (SD), conditions in which pathogenesis-related responses were not activated in cat2, the additional presence of the pp2a-b′γ mutation allowed lesion formation, PATHOGENESIS-RELATED GENE1 (PR1) induction, salicylic acid (SA) and phytoalexin accumulation and the establishment of metabolite profiles that were otherwise observed in cat2 only in LD. Lesion formation in cat2 pp2a-b′γ in SD was genetically dependent on SA synthesis, and was associated with decreased PHYTOCHROME A transcripts. Phosphoproteomic analyses revealed that several potential protein targets accumulated in the double mutant, including recognized players in pathogenesis and key enzymes of primary metabolism.
  • We conclude that the cat2 and pp2a-b′γ mutations interact synergistically, and that PP2A-B′γ is an important player in controlling day length-dependent responses to intracellular oxidative stress, possibly through phytochrome-linked pathways.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Modifications of the cellular redox state associated with oxidative stress are an integral part of plant responses to the environment (Apel & Hirt, 2004; Van Breusegem et al., 2008; Foyer & Noctor, 2009). Although increased availability of reactive oxygen species (ROS) can induce malfunction through oxidative modification of sensitive cell components, the phenotypic and physiological outcomes of oxidative stress are mediated by close interactions with signaling through phytohormones, such as salicylic acid (SA), jasmonic acid and ethylene (Overmyer et al., 2003). Because photosynthetic processes, such as chloroplastic electron transport and photorespiration, can be major sources of ROS, the intensity of oxidative stress may be modified by light intensity (irradiance). In addition, growth day length is emerging as a key factor that may modulate ROS availability or, downstream of ROS accumulation, the response to oxidative stress (Bechtold et al., 2005; Becker et al., 2006; Queval et al., 2007, 2012; Vollsnes et al., 2009; Michelet & Krieger-Liszkay, 2011; Kangasjärvi et al., 2012; Dghim et al., 2013; Pérez-Pérez et al., 2013). Because of the centrality of oxidative stress in plant responses to external challenges, an understanding of the underlying factors is potentially relevant to efforts to predict and manipulate stress resistance in the natural environment, where day length is a crucial modulator of plant growth and development.

The Arabidopsis mutant cat2, which lacks the major leaf catalase, is a useful study system for the identification of factors involved in the response to hydrogen peroxide (H2O2) produced inside the cell through a physiologically relevant pathway (Mhamdi et al., 2010a). Because CAT2 function is closely linked to photorespiration, loss of its function triggers oxidative stress in plants grown under moderate irradiance in air, when glycolate-dependent H2O2 production in the peroxisomes is significant. When grown in these conditions in long days (LD), cat2 shows decreased growth accompanied by many of the features shown by lesion mimic mutants. This includes many pathogenesis-related (PR) responses, including lesion formation, accumulation of SA and phytoalexins, expression of PR genes and induced resistance to bacterial challenge (Chaouch et al., 2010). All of these effects, apart from decreased growth, are annulled in cat2 sid2 double mutants, in which SA production through the isochorismate pathway is blocked (Chaouch et al., 2010). Similar decreases in growth in cat2 sid2 and cat2, as well as the responses of biochemical and transcriptomic markers of the redox state, show that cat2-triggered intracellular oxidative stress and SA-dependent responses can be uncoupled.

Responses to oxidative stress in cat2 are strongly influenced by day length. When the mutant is grown under moderate or even high light in short days (SD), oxidative stress is apparent, but lesions are absent and SA-dependent responses are not activated (Queval et al., 2007; Chaouch et al., 2010). Moreover, distinct transcriptomic and metabolomic signatures are observed in cat2 grown in SD or LD conditions (Queval et al., 2007, 2012; Chaouch et al., 2010). The conditional day length-dependent nature of the oxidative stress responses in cat2 implies that genetic factors interact with the environment to govern responses to oxidative stress.

Genetic factors determine cell death triggered by excess singlet oxygen generation in the fluorescent (flu) mutant (Wagner et al., 2004). The SA-dependent lesion phenotype observed in cat2 in LD is also strongly modulated by secondary mutations, including several that cause loss of function of recognized players in pathogenesis pathways, such as NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and Arabidopsis thaliana respiratory burst oxidase homolog F (AtRbohF) (Chaouch et al., 2010, 2012; Han et al., 2013a,b). It is also affected by genetically blocking glutathione accumulation and by mutations in certain NADPH-producing enzymes (Mhamdi et al., 2010b; Han et al., 2013a,b). Despite the clear effect of these secondary mutations on cat2 responses in LD, we have identified none that permit oxidative stress to trigger lesions and related responses in cat2 grown in SD conditions. The factors that repress oxidative stress-triggered pathogenesis responses in SD conditions therefore remain unknown.

Although much remains to be elucidated on the link between oxidative stress and pathogenesis responses, protein phosphorylation status is involved (Kovtun et al., 2000; Apel & Hirt, 2004). Type 2A protein phosphatases (PP2A) account for a significant part of total protein phosphatase activity in plant tissue extracts (MacKintosh & Cohen, 1989). These enzymes are composed of three types of subunit (A, B, C), with each subunit being encoded by several genes. Theoretically, the interaction of A, B and C subunits encoded by the different genes could produce 255 possible heterotrimeric combinations, allowing a potentially wide range of specific functions in protein dephosphorylation (Zhou et al., 2004). Genes encoding the regulatory B subunit are the most numerous (17 genes, subclassified into B, B′ and B″), probably reflecting the importance of these polypeptides in determining the substrate specificities and physiological functions of the different PP2A holoenzymes. Recently, novel roles for a gene encoding a specific PP2A-B′ subunit have been described (Trotta et al., 2011a,b). Loss-of-function mutants for PP2A-B′γ were shown to constitutively activate pathogenesis responses (Trotta et al., 2011a). However, how this PP2A subunit functionally interacts with ROS and the cellular redox state remains unknown. Outstanding questions include: (1) whether PP2A-B′γ interacts with day length to influence oxidative stress outcomes; (2) the proteins that may be affected by PP2A-B′γ during any such function; (3) the influence of PP2A-B′γ in oxidative stress-triggered modulation of plant metabolism; and (4) whether this subunit plays a role in signaling upstream or downstream of intracellular oxidative stress.

Through a genetics-based analysis of the interaction between intracellular oxidative stress and PP2A-B′γ, this study provides new information on these questions. Most notably, we show that the repression of SA-dependent PR responses when oxidative stress is occurring in SD is dependent on PP2A-B′γ, and report novel proteomics data on PP2A-B′γ-sensitive phosphorylation targets during intracellular oxidative stress.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material

The species studied was Arabidopsis thaliana (L.) Heynh T-DNA, and other mutant lines were all generated in the Col-0 background. The cat2 line was cat2-1 (Queval et al., 2009) and the pp2a-b′γ mutant was SALK_039172 (Trotta et al., 2011a). The cat2 pp2a-b′γ line was produced by crossing, and triple mutants were obtained by crossing the double mutant with cat2 sid2 or cat2 npr1, lines that have been described previously (Chaouch et al., 2010; Han et al., 2013a).

Identification of homozygous double and triple mutants

After verification of double heterozygotes in the F1 generation by PCR, double and triple homozygotes were identified similarly in plants grown from F2 seeds. For T-DNA insertions, leaf DNA was amplified by PCR (30 s at 94°C, 30 s at 60°C, 1 min at 72°C, 30 cycles) using primers specific for left T-DNA borders and the CAT2 and PP2A-B′γ genes (Supporting Information Table S1). Zygosity of the SA induction-deficient 2 (sid2) and npr1 mutations was established using restriction length polymorphism (Fig. S1). Functional analyses were performed on plants grown from F3 seeds.

Plant growth and sampling

Unless indicated otherwise, plants were grown in a controlled environment growth room under a day : night regime of 8 h : 16 h (SD) or 16 h : 8 h (LD), an irradiance of 200 μmol m−2 s−1 at leaf level, temperatures of 20°C day : 18°C night, 65% humidity and a CO2 concentration of 400 μl l−1. Nutrient solution was supplied twice per week. Plants were analyzed and sampled at the age of 21–24 d (LD) and 35 d (SD). For complementation experiments, plants were grown in SD for 5 wk, and 10 mM myo-inositol was sprayed onto the rosettes daily from the fourth week onwards. Samples were taken in the middle of the photoperiod (n = 3–5 independent biological replicates), rapidly frozen in liquid nitrogen and stored at −80°C. The percentage lesion areas on the leaves were quantified using IQmaterials software. Significant differences are expressed using Student's t-test at P < 0.05. All experiments were repeated at least twice with similar results. Staining with 3,3′-diaminobenzidine (DAB) was performed as in Mhamdi et al. (2010c).

Quantitative PCR (qPCR) analysis

RNA was extracted using the TRIZOL reagent (Life Technologies, Saint Aubin, France). cDNA was produced by reverse transcription using the SuperScript™ III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). qPCR was performed as in Queval et al. (2007). Primer sequences are listed in Table S1.

Metabolite profiling

Targeted analysis of antioxidants was performed by plate-reader (Queval & Noctor, 2007). SA, scopoletin and camalexin were measured by high-performance liquid chromatography (HPLC)-fluorescence as in Langlois-Meurinne et al. (2005). Non-targeted metabolite profiling by gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) followed previously described protocols (Noctor et al., 2007; Chaouch et al., 2012). Details are provided in Table S2(c). Peak deconvolution and integration of specific ions were performed using the LECO Pegasus software, based on mass spectra available in the National Institute of Standards and Technology (NIST) and MPI-GOLM reference libraries, as well as an in-house database generated using authentic standards. To produce the heatmap display, values were centered and reduced. For each compound, means and standard deviations were calculated across all samples. For each genotype, the mean value for compound ‘x′ was subtracted from the mean across all samples, and the value was then divided by the overall standard deviation for that compound.

Proteomics

Extraction of total foliar extracts and total soluble leaf extracts was performed in the presence of protease (Complete-Mini; Roche) and phosphatase (PhosSTOP; Roche) inhibitors using the methods described by Kangasjärvi et al. (2008). Total soluble leaf extracts and membrane fractions corresponding to 80 μg of proteins were used for mono-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or CN-PAGE (clear-native PAGE) followed by SDS-PAGE. Soluble oligomeric protein complexes were separated by CN-PAGE followed by SDS-PAGE in the second dimension according to Peltier et al. (2006) with the modifications for buffers described by Rokka et al. (2005), except that only 0.05% deoxycholate was used as a detergent on CN gels. After imaging the gels with Sypro Ruby (Invitrogen), MS was performed as described by Peltier et al. (2006) using a liquid chromatography-electrospray ionization-MS/MS system (Q-Exactive; Thermo Fisher Scientific, Waltham, MA, USA, and QTOF Elite; AB Sciex, Framingham, MA, USA). MS/MS spectra were analyzed with an in-house installation of Mascot (www.matrixscience.com), with searches restricted to the Uniprot Arabidopsis database. Results were analyzed through Proteome Discover v.1.3 (Thermo Scientific) allowing methionine (Met) oxidation, cysteine (Cys) carboamidomethylation and serine/threonine (Ser/Thr) and tyrosine (Tyr) phosphorylation as possible modifications, and validating the phosphopeptides through PhosphoRS filter and Decoy Database Search, with target false discovery rates (FDR) of < 0.01 (strict) and < 0.05 (relaxed). The confidence threshold for phosphopeptides was set to < 0.05. Phosphopeptides above this threshold were validated manually.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The pp2a-b′γ mutant has been characterized and described previously by Trotta et al. (2011a). To explore interactions between intracellular oxidative stress and PP2A-B′γ function, this line was crossed with cat2 to produce double mutants, and the interplay between the two mutations in determining phenotypes, defense and metabolic responses was examined in LD (16 h photoperiod) and SD (8 h photoperiod) conditions.

Interactions between cat2 and pp2a-b′γ mutations during growth in LD

First, plants were analyzed by following growth in LD at moderate light, conditions in which pathogenesis responses are activated by oxidative stress in cat2. All three mutant lines were smaller than the wild-type, with the double mutant showing a significant decrease relative to cat2 (Fig. 1a). Lesions were clearly apparent in both cat2 backgrounds, and slightly more extensive in cat2 pp2a-b′γ. The pp2a-b′γ single mutant also presented some lesions, although to a much lesser extent than cat2 (Fig. 1a). There was an overall correlation between lesion extent and expression of PATHOGENESIS-RELATED GENE1 (PR1). This SA marker gene was strongly induced in cat2, but not significantly in pp2a-b′γ (Fig. 1b). In the double mutant, PR1 expression was increased to about two-fold the cat2 values (Fig. 1b). Analysis of SA and related defense compounds showed a similar pattern, with some induction in the pp2a-b′γ mutant and stronger induction in cat2. The double mutant generally showed the most marked accumulation of these compounds, and this was particularly apparent for camalexin, a major phytoalexin in Arabidopsis (Glawischnig, 2007), which was about three-fold higher in cat2 pp2a-b′γ than in cat2 (Fig. 1c). Together, these observations show that loss of PP2A-B′γ function produces the activation of defense responses, as reported previously (Trotta et al., 2011a), and that the mutation reinforces these responses in cat2.

image

Figure 1. Growth and pathogenesis-related responses in Arabidopsis Col-0, cat2, pp2a-b′γ and cat2 pp2a-b′γ growing in long days (16 h light : 8 h dark). (a) Representative photographs of plants and quantification of rosette mass and lesion area. (b) PATHOGENESIS-RELATED GENE1 (PR1) expression measured by quantitative PCR (qPCR). (c) Salicylic acid (SA) and related defense compounds. Data are means ± SE of at least 10 plants (a) or three to four biological replicates (b, c). * and +, significant difference relative to Col-0 or to cat2 (< 0.05). FW, fresh weight; nd, not detected.

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In our hands, changes in H2O2 are negligible in cat2, either because of rapid metabolism by alternative pathways, technical difficulties in the specific quantification of this reactive molecule, or both (Queval et al., 2008; Mhamdi et al., 2010a,c). Increased accumulation of oxidized glutathione is a well-described response in catalase-deficient plants (Smith et al., 1984; Willekens et al., 1997), and is the clearest biochemical marker of oxidative stress in cat2 (Queval et al., 2007; Mhamdi et al., 2010a,c). This key determinant of intracellular thiol-disulfide status is clearly implicated in pathogenesis and other plant stress responses (Ball et al., 2004; Parisy et al., 2007), and is functionally important in determining the activation of phytohormone signaling and related phenotypes in cat2 grown in LD (Han et al., 2013a,b). Like glutathione, ascorbate is a marker of tissue redox status, and has been implicated in responses to biotic stress (Pastori et al., 2003). The reduced and oxidized forms of these two molecules were therefore quantified as markers of cellular redox state.

In all mutants, ascorbate was slightly more oxidized than in Col-0, but, in agreement with previous studies, its status was much less affected than glutathione by the cat2 mutation (Fig. S2). In cat2, glutathione was increased c. 2.5-fold, and this was associated with a drop in the redox state from over 90% to c. 60% reduced (Fig. S2). Slight oxidation of glutathione was also observed in pp2a-b′γ, but the combination of the two mutations did not cause further oxidation or accumulation relative to cat2 (Fig. S2).

One feature of the cat2 mutant is its conditional photorespiratory nature. This means that this line shows little sign of oxidative stress when grown at high CO2 or low light, conditions in which photorespiration is negligible or slow. At low light, the pp2a-b′γ mutant showed a similar decrease in growth to that observed at moderate light (Fig. S3). By contrast, the impact of the cat2 mutation on rosette size or glutathione status was much less marked than at the standard irradiance (compare data in Figs 1 and S3). In low light conditions, no lesions were detectable on the leaves of any of the mutants. Experiments in which plants were grown at high CO2 also revealed that no lesions were observed on single or double mutants (data not shown). Thus, whether or not PP2A-B′γ was functional, lesions triggered by the cat2 mutation were conditionally dependent on a threshold rate of photorespiratory H2O2 production, and the oxidative stress that resulted from it.

Effect of the pp2a-b′γ mutation in SD, conditions that are non-permissive for the activation of pathogenesis responses in cat2

Because the above data reveal a functional interaction between the pp2a-b′γ and cat2 mutations on the pathogenesis responses in LD, we focused the remainder of our study on establishing whether PP2A-B′γ could be involved in regulating these responses in cat2 grown in SD. When plants were grown in SD at the same irradiance as in LD (Fig. 1), oxidative stress in cat2 produced a decrease in growth, but no lesions were observed and PR1 was not induced (Fig. 2). Lesions were also absent on the leaves of pp2a-b′γ. By contrast, the cat2 pp2a-b′γ double mutant showed significant lesion formation and induction of PR1 (Fig. 2). Unlike cat2, the double mutant also showed significant DAB staining (Fig. 2).

image

Figure 2. The pp2a-b′γ mutation allows intracellular oxidative stress to trigger lesions and PATHOGENESIS-RELATED GENE1 (PR1) expression responses in Arabidopsis in otherwise non-permissive conditions (short days; 8 h light : 16 h dark). Data are means ± SE of at least 10 plants (lesions) or five biological replicates (PR1). Photographs at the bottom show staining of tissues with 3,3′-diaminobenzidine (DAB). Note that the top and bottom series of photographs show different plants; nd, not detected.

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Redox analysis revealed that the appearance of lesions in the double mutant was not associated with marked increases in oxidation of either ascorbate or glutathione relative to cat2 (Fig. 3). Similar to effects observed in LD (Fig. S2), the double mutant showed a slight tendency to increased oxidation of ascorbate (Fig. 3). The pp2a-b′γ mutation did not affect glutathione status in the Col-0 background. In cat2 in SD, glutathione was even more oxidized than in LD (Fig. 3; compare with data in Fig. S2), but neither the oxidation state nor the accumulation was further increased by the secondary pp2a-b′γ mutation. Indeed, the lesions observed in SD in the double mutant (Fig. 2) were associated with a more reduced glutathione pool than in cat2 (Fig. 3), in which no lesions were apparent in this condition (Fig. 2).

image

Figure 3. Major leaf antioxidant pools in Arabidopsis Col-0, cat2, pp2a-b′γ and cat2 pp2a-b′γ growing in short days. White bars, reduced forms; black bars, oxidized forms. Data are means ± SE of three biological replicates. Where significant, * and + indicate difference relative to Col-0 or to cat2 (< 0.05) for each form. Numbers above the bars indicate the percentage of ascorbate or glutathione in the reduced form. FW, fresh weight.

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To identify the metabolic changes underlying the lesions induced in cat2 pp2a-b′γ in SD, non-targeted profiling of almost 100 metabolites was performed by GC-TOF-MS (Table S2). Using this technique, we have previously identified a specific metabolite signature induced by intracellular oxidative stress in an LD- and SA-dependent manner (Chaouch et al., 2010). This signature shows considerable overlap with that induced by bacterial challenge, and its main features are absent in both cat2 grown in SD and in a cat2 sid2 mutant in LD (Chaouch et al., 2010, 2012). Hierarchical clustering of all metabolites quantified in samples taken in SD revealed that the single cat2 and pp2a-b′γ mutations had only a minor impact (Fig. 4, left). By contrast, a marked effect on metabolite profiles was observed in the double mutant (Fig. 4, left). This involved the accumulation of a large subcluster of compounds that accumulated much less or not at all in the cat2 single mutant. Comparison with a previously described dataset revealed that most of these compounds were those that accumulated in the cat2 single mutant in LD, but not in cat2 sid2 (Fig. 4). Thus, the pp2a-b′γ mutation allows intracellular oxidative stress to induce metabolic responses in SD that are only observed in LD when PP2A-B′γ is functional, and these responses are dependent on the isochorismate pathway of SA synthesis. Indeed, SA itself was among the metabolites that were most clearly dependent on the cat2pp2a-b′γ interaction (Fig. 4). Others included nicotinic acid, which has been implicated in pathogenesis responses (Noctor et al., 2011), and gluconic acid, which is strongly induced by bacterial challenge as well as in cat2 in LD (Chaouch et al., 2012; Tables 1, S2).

Table 1. The most induced metabolites in the leaves of the Arabidopsis cat2 pp2a-b′γ double mutant in short days (SD). Fifty-three significantly different metabolites were identified in cat2 pp2a-b′γ relative to Col-0 (Table S2b). The 10 most induced of these are shown below
MetaboliteFold changes
Short days (this study)Long days (Chaouch et al., 2012)
cat2 cat2 pp2a-b′γcat2 pp2a-b′γ cat2
rel. Col-0rel. Col-0rel. cat2rel. Col-0
  1. a

    Significant difference at ≤ 0.05. Values are shown for cat2 SD and cat2 pp2a-b′γ SD (this study) and cat2 LD (Chaouch et al., 2012). All values are relative to the corresponding Col-0 values. ND, metabolite not detected in earlier study; + indicates metabolites induced in cat2 in LD, but not detectable in Col-0; therefore, the fold change cannot be calculated.

Nicotinic acid1.095.1a94.0a3.9
Gluconic acid26.7a53.9a2.0a95.9
Norvaline13.0a17.8a1.4ND
Aminoadipic acid1.012.2a12.0a+
2-Hydroxyglutaric acid1.010.8a10.6a13.6
Arginine1.710.5a6.1a5.4
Tryptophan1.09.15a9.0a+
Salicylic acid1.07.6a7.5a7.2
Glucose 6-phosphate6.2a7.6a1.2ND
Cysteine2.6a5.4a2.1a9.1
image

Figure 4. Gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) analysis of Arabidopsis leaves reveals that the pp2a-b′γ mutation allows the induction of cat2 metabolite profiles in short days that are only otherwise induced in long days in an ISOCHORISMATE SYNTHASE1 (ICS1)-dependent manner. Left heatmap, display of identified metabolites in the four plant lines grown in short days. Right heatmap, corresponding data from Chaouch et al. (2010), replotted in a new form, for Col-0, cat2 and cat2 sid2 grown in long days. White rows indicate metabolites that were not detected in at least one sample in the previous analysis. Graphs on the right show selected metabolites that are induced in cat2 pp2a-b′γ in short days, with a comparison of their response in cat2 and cat2 sid2 in long days (means ± SE of three biological replicates).

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The combined presence of the two mutations produced striking changes in many amino acids. In our conditions, although minor increases in homoserine and tryptophan were apparent, the pp2a-b′γ single mutant showed amino acid profiles that were similar to those of the wild-type (Fig. S4). When introduced into the cat2 background, the pp2a-b′γ mutation markedly stimulated amino acid accumulation. In some cases, the main effect was to reinforce increases that were already evident in cat2 (e.g. glutamate, glutamine, Cys, Met, Ser, O-acetylserine). In many cases, however, the two mutations acted together to produce increases that were minor or negligible in the single mutants. Striking examples of compounds that responded in this way were isoleucine, ornithine, Thr, lysine, β-alanine, GABA, phenylalanine, Tyr, tryptophan and arginine (Fig. S4), the last two featuring among the most strongly induced significant metabolites observed in cat2 pp2a-b′γ (Table 1).

To further investigate the role of SA, this compound was quantified by HPLC in plants grown in SD, together with the phytoalexin, camalexin. Despite the evident disulfide stress in cat2 (Fig. 3), the amounts of SA and camalexin were not significantly different from those in Col-0 (Fig. 5). Some induction of SA was observed in pp2a-b′γ, but the accumulation was much more marked in the double mutant, where total SA reached levels observed in cat2 in LD (Fig. 1). Camalexin accumulation above basal Col-0 levels was only significant in cat2 pp2a-b′γ, in which levels of this compound were similar to those in cat2 in LD (Fig 5; compare with Fig. 1). To establish whether the lesions associated with pathogenesis responses in cat2 pp2a-b′γ in SD were dependent on the SA pathway, triple mutants were produced that additionally carried either sid2, defective in SA synthesis (Wildermuth et al., 2001), or npr1, defective in SA signaling (Cao et al., 1994). In cat2 pp2a-b′γ npr1, lesions were significantly decreased, whereas, in cat2 pp2a-b′γ sid2, they were absent (Fig. 6). As reported previously for cat2 growing in LD, lesion formation in cat2 pp2a-b′γ in SD could be prevented simply by treating plants with myo-inositol (Fig. 6), a compound that is implicated in the SA signaling pathway (Meng et al., 2009; Chaouch & Noctor, 2010; Donahue et al., 2010). Together, these results show that the lesions produced in cat2 pp2a-b′γ in SD were dependent on the SA pathway in a similar manner to the lesions observed in the cat2 single mutant in LD.

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Figure 5. Loss of PP2A-B′γ function allows the cat2 mutation to trigger the accumulation of salicylic acid (SA) and camalexin in Arabidopsis growing in short days. Data are means ± SE of four biological replicates. * and +, significant difference relative to Col-0 or to cat2 (< 0.05). FW, fresh weight.

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image

Figure 6. Complete or partial genetic reversion of the Arabidopsis cat2 pp2a-b′γ lesion phenotype in short days by sid2 and npr1 mutations, and chemical blocking of the lesions by myo-inositol (MI) treatment. For lesion quantification, data are means ± SE (n = at least 10 plants). Examples of lesion-forming areas are indicated on the photographs by white arrows. nd, not detected.

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Proteome and phosphoproteome modifications induced by the pp2a-b′γ mutation during oxidative stress

Next, we assessed how the features related to defense signaling were reflected in the proteome, and particularly the phosphoproteome, of cat2 pp2a-b′γ double mutant plants. First, we analyzed the pattern of phosphoproteins in total soluble and membrane fractions isolated from leaves of Col-0, cat2, pp2a-b′γ and cat2 pp2a-b′γ grown in SD. SDS-PAGE and subsequent quantitative ProQ staining of phosphoproteins, followed by Sypro staining of total proteins, showed no major changes between the cat2 and pp2a-b′γ single mutants and Col-0 (Fig. S5). By contrast, a set of highly phosphorylated bands emerged in the soluble proteome of cat2 pp2a-b′γ double mutants (Fig. S5). None of the genotypes showed any major changes in the membrane fractions (Fig. S5).

Because neither single mutant showed marked changes in protein profiles, two-dimensional gel analysis was used to identify the phosphoproteins observed in cat2 pp2a-b′γ. This was performed in two successive steps. First, oligomeric protein complexes in total soluble leaf extracts were separated by CN-PAGE, followed by SDS-PAGE in the second dimension (Fig. 7), and stained with ProQ followed by Sypro. To detect the phosphopeptides, the gels were silver stained and the spots of interest were identified by MS and analysis of MS/MS spectra, allowing Ser, Thr and Tyr phosphorylation as possible modifications (Table 2; see Table S3 for technical details).

Table 2. Summary of proteins and phosphopeptides identified in soluble protein extracts of Arabidopsis leaves by two-dimensional clear-native polyacrylamide gel electrophoresis (CN-PAGE) and tandem mass spectrometry (MS/MS) analysis. Where corresponding phosphopeptides were detected, these are indicated in the right columns by ‘X’, and the peptide sequence containing the phosphorylated amino acid is given to the left.
Spot ID and change in cat2 pp2a-b′γaFull nameGenePhosphopeptidesbCol-0cat2 pp2a-b′γ
  1. a

    The spot identifications are as presented in Fig. 7. Protein spots showing higher or lower intensity relative to the wild-type are indicated by upward or downward arrows, respectively.

  2. b

    For the identification of phosphopeptides, Mascot analysis of MS/MS spectra was conducted by restricting searches for the Arabidopsis database and allowing the phosphorylation of threonine (Thr), serine (Ser) and tyrosine (Tyr) residues as possible modifications. Details on the identifications are presented in Table S3.

  3. c

    This peptide is unique to At4g13940, whereas the others are identical for both isoforms.

  4. d

    For these peptides, phosphoRS analysis was unable to indicate with high probability which residue is phosphorylated.

Frame (c)
1 [UPWARDS ARROW]Protein disulfide isomerase 2At5g60640VE(p)TTETKESPDSTTK X
2 [DOWNWARDS ARROW]Catalase 2At4g35090LGPN(p)YLQLPVNAPKX 
3 [UPWARDS ARROW]S-Adenosyl-l-homocysteine hydrolase 1At4g13940(p)TEFGPSQPFKGARcXX
(p)SKFDNLYGCRXX
S-Adenosyl-l-homocysteine hydrolase 2At3g23810LVGV(p)SEETTTGVKXX
GE(p)TLQEYWWCTER X
4 [UPWARDS ARROW]ADP glucose pyrophosphorylase large subunit 1At5g19220V(P)YILTQYNSASLNRX 
(p)TVASIILGGGAGTRXX
5 [UPWARDS ARROW]Calreticulin-1At1g56340   
6 [UPWARDS ARROW]Calreticulin-2At1g09210LL(p)SGDVDQKK X
7 [UPWARDS ARROW]Elongation factor 1-αAt1g07940G(p)YVASNSKDDPAKXX
MTPTKPMVVE(p)TFSEYPPLGRX 
MTP(p)TKPMVVETFSEYPPLGR X
(p)STTTGHLIYKdX 
(p)YYCTVIDAPGHR X
VETGMIKPGMVV(p)TFAPTGLTTEVK X
Frame (d)
1 [UPWARDS ARROW]Glutamine synthetase 1;1At5g37600(p)TLPGPVTDPSQLPK X
2 [DOWNWARDS ARROW]Serine:glyoxylate aminotransferaseAt2g13360L(p)SQDENHTIKXX
(p)TLLEDVKKXX
AL(p)SLPTGLGIVCASPKX 
AICIVHNE(p)TATGVTNDISAVRX 
Hydroxypyruvate reductaseAt1g68010EGMA(p)TLAALNVLGRXX
A(p)SSMEEVLRX 
3 [UPWARDS ARROW]Monodehydroascorbate reductaseAt5g03630   
4 [UPWARDS ARROW]β-1,3-Glucanase2, pathogenesis-related protein 2At3g57260T(p)YVNNLIQHVK X
LA(p)SSQTEADKWVQENVQSYRd X
MRL(p)YGPDPGALAALR X
ACC oxidase 2At1g62380NASAVTELNPTAAVE(p)TF X
HLPQ(p)SNLNDISDVSDEYR X
5 [UPWARDS ARROW]31-kDa RNA binding proteinAt4g24770   
3-β-Hydroxysteroid dehydrogenase domain-containing proteinAt2g37660ALF(p)TQVTTKFX 
(p)TGQIVYKKXX
6 [UPWARDS ARROW]HSP20-likeAt4g02450APAAEE(p)TTSVKEDKXX
Thiocyanate methyltransferase 1At2g43910AV(p)SVEENPHAIPTRXX
A(p)TPLIVHLVDTSSLPLGR X
7 [UPWARDS ARROW]Pathogenesis-related gene 5At1g75040YAGCV(p)SDLNAACPDMLK X
NNCP(p)TTVWAGTLAGQGPK X
Frame (e)
1 [DOWNWARDS ARROW]Plastid isoform triose phosphate isomeraseAt2g21170EAGK(p)TFDVCFAQLKXX
NV(p)SEEVASKTRXX
VA(p)SPQQAQEVHVAVRXX
GPEFA(p)TIVNSVTSKKX 
2Glutathione S-transferase TAU 20At1g78370FGNF(p)SIESESPKXX
(p)SPLLLQSNPIHKXX
(p)SLPDSEKIVAYAAEYRX 
3[DOWNWARDS ARROW]Carbonic anhydrase1At3g01500(p)YMVFACSDSRX 
(p)YETNPALYGELAKX 
YE(p)TNPALYGELAKGQSPKXX
EK(p)YETNPALYGELAKXX
EKYETNPAL(p)YGELAKXX
EKYE(p)TNPALYGELAK X
G(p)TLALKGGYYDFVKXX
VI(p)SELGDSAFEDQCGRXX
VCP(p)SHVLDFQPGDAFVVRXX
4Glutathione S-transferase U19At1g78380GVEFEYREEDLRXX
V(P)TEFVSELRKXX
(p)SPLLLQMNPIHKX 
5 [UPWARDS ARROW]Copper/zinc superoxide dismutaseAt2g28190   
6 [UPWARDS ARROW]Glutathione S-transferase PHI 2At4g02520AI(p)TQYIAHR X
(p)SIYGLTTDEAVVAEEEAK X
(p)YENQGTNLLQTDSK X
image

Figure 7. Proteomic analysis of leaves of the Arabidopsis cat2 pp2a-b′γ double mutant growing in short days. (a, b) Representative Sypro-stained (a) and ProQ-stained (b) two-dimensional gels depicting proteins in the total foliar soluble fractions isolated from wild-type Col-0 and cat2 pp2a-b′γ plants. Oligomeric protein complexes were separated by clear-native polyacrylamide gel electrophoresis (CN-PAGE), followed by sodium dodecylsulfate (SDS)-PAGE in the second dimension. (c–e) Magnified frames from ProQ- and Sypro-stained gels showing areas with protein spots of higher or lower intensity in cat2 pp2a-b′γ relative to Col-0. The protein spots identified are indicated by sequential numbers in each frame, with each number indicating the same protein in cat2 pp2a-b′γ and Col-0. The proteins were identified by in-gel trypsin digestion and subsequent analysis by a nano-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoHPLC-ESI-MS/MS) system. Details concerning the identified proteins and phosphopeptides are presented in Table 2.

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A number of spots with altered protein and/or phosphopeptide abundance between cat2 pp2a-b′γ and wild-type plants were observed (Fig. 7, Table 2). Proteins with different abundance included S-adenosyl-l-homocysteine hydrolase (SAHH) (spot 3, frame c), glutamine synthetase 1;1 (GLN1;1) and serine:glyoxylate aminotransferase (SGAT)/hydroxypyruvate reductase (spots 1 and 2, frame d), reflecting the marked changes observed in amino acid metabolism (Fig. S5), as well as copper/zinc superoxide dismutase (CSD2) and glutathione S-transferase PHI 2 (GSTF2) (spot 5 and 6, frame e), related to cellular redox homeostasis (Fig. 7, Table 2). These proteins have also been found previously to be affected in the pp2a-b′γ single mutant grown under different light and humidity conditions (130 μmol photons m−2 s−1, 50% relative humidity; Trotta et al., 2011a,b). The two-dimensional gel approach also identified a distinct spot containing AtMDAR2 (Arabidopsis thaliana monodehydroascorbate reductase2) (spot 3, frame d) in cat2 pp2a-b′γ, whereas, in the wild-type, this spot was barely visible (Fig. 7). In agreement with increased PR1 transcripts (Fig. 2) and SA contents (Fig. 5), the double mutant also showed a marked accumulation of the SA-dependent proteins, PR2 and PR5 (spots 4 and 7, frame d). PR2 co-migrated with ACC oxidase 2 (ACO2), an ethylene-forming enzyme, which, like PR2, was not visible in Col-0 extracts (Fig. 7, Table 2).

With the exception of AtMDAR2, all the above-mentioned proteins contained phosphorylated peptides, and were thus identified as phosphoproteins (Table 2). Phosphopeptides specific for cat2 pp2a-b′γ were observed in SAHH and GSTF2, as well as in GLN1;1, PR2, PR5 and ACO2, proteins that were detectable only in the mutant extracts (Fig. 7, Table 2). SGAT, by contrast, contained two phosphopeptides that were detected in the wild-type, but not in cat2 pp2a-b′γ (Table 2).

Among the proteins recognized by ProQ, protein disulfide isomerase 2 (PDI2), Calreticulin 1 (CRT1) and CRT2 (spots 1, 5 and 6, respectively, frame c) showed a marked up-regulation of the protein level in cat2 pp2a-b′γ compared with wild-type plants (Fig. 7). MS revealed no phosphorylated residues in CRT1, whereas PDI1 and CRT2 contained phosphorylated peptides that were present in cat2 pp2a-b′γ, but not in the wild-type (Table 2). An increased phosphorylation level of elongation factor 1-α (EF1α) was also observed, indicative of regulatory changes at the level of protein synthesis in the cytosol as well (Fig. 7, Table 2).

The starch metabolism enzyme ADP glucose pyrophosphorylase large subunit 1 (AGPase) (spot 4, frame c) and an unknown protein At2g37660 (spot 5, frame d) were also detected by ProQ staining, but these proteins contained less phosphopeptides in cat2 pp2a-b′γ than in wild-type plants (Fig. 7, Table 2). In the intensively Sypro-stained spot 5 in frame (d), the protein with highest abundance was a chloroplastic 31-kDa RNA binding protein, but for which no phosphopeptides could be identified (Table 2). Spot 6 in frame (d) contained two phosphoproteins, HSP20-like protein and thiocyanate methyltransferase 1 (HOL1). Our previous study identified the myrosinase TGG1, a glucosinolate-degrading enzyme, as a distinctly up-regulated spot in pp2a-b′γ (Trotta et al., 2011a), and this spot was evident in cat2 pp2a-b′γ (Fig. 7).

Two spots, containing a plastid isoform of triose phosphate isomerase (PDTPI) (spot 1, frame e) and carbonic anhydrase1 (CA1) (spot 3, frame e), appeared to be clearly down-regulated and contained less phosphopeptides in cat2 pp2a-b′γ relative to wild-type plants (Table 2). Although CA1 has been implicated in SA-dependent responses, the triose phosphate isomerase catalyzes the conversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate in the Benson–Calvin cycle.

Finally, we also found that CAT2 co-migrated as a high-molecular-weight oligomeric protein complex in the soluble leaf extract of wild-type plants, and also contained a phosphorylated Tyr residue at the C-terminal part of the protein (Fig. 7, spot 2, frame c). In cat2 pp2a-b′γ, the disappearance of CAT2 coincided with a decrease in the co-migrating SGAT (Fig. 7).

To determine whether the increased abundance detected at the protein level was accompanied by similar changes in transcript abundance, qPCR analysis of selected genes was performed. In agreement with the proteomics data (Table 2), GLN1;1 and GSTF2 transcripts were much more abundant in the double mutant than in other genotypes (Fig. 8a). This pattern was not observed for the transcripts of three other proteins found to accumulate in cat2 pp2a-b′γ (Fig. 8a), suggesting that their increased abundance could be related to regulation at the post-transcriptional level.

image

Figure 8. Quantification of transcripts encoding differentially abundant proteins identified on the two-dimensional gels (a) and genes involved in flowering control or photoperiod signaling (b). Data are for Arabidopsis grown in short days. For explanation of abbreviations, see Supporting Information Table S1. Data are means ± SE of three biological replicates. For ease of expression, FT and CO values have been multiplied by 10 000, and FLC and PHY values by 100. Where significant, * and + indicate difference relative to Col-0 or to cat2 (< 0.05).

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Flowering time and related gene expression in the different genotypes

Photoreceptors and regulation of flowering are among the possible pathways by which the PP2A-B′γ-dependent processes might mediate day length regulation of oxidative stress responses. This possibility was explored in two ways. First, an analysis was undertaken of the time taken to produce flowering stems in LD conditions, which promote the flowering program in Arabidopsis. No marked difference was observed between Col-0 and cat2, but the flowering stem appeared later in both the pp2a-b′γ single mutant and cat2 pp2a-b′γ than in Col-0 and cat2 (Fig. S6). Delayed flowering is in agreement with effects reported for the pp2a-b′γ mutant in a recent study of the role of PP2A subunits in flowering regulation (Heidari et al., 2013). Second, the expression levels of three genes involved in the control of flowering (CONSTANS (CO), FLOWERING LOCUS T (FLT), FLOWERING LOCUS C (FLC)) were determined in SD, conditions in which the pp2a-b′γ mutant plays an important role in controlling the outcome of oxidative stress. This analysis revealed very low expression in all genotypes, and little or only slight differences between them (Fig. 8b). Slightly higher levels of FLC expression were observed in the pp2a-b′γ genotypes, although this was only significant for cat2 pp2a-b′γ (Fig. 8b). These increases were much less apparent than increases in FLC expression reported in pp2a-b′γ in LD (Heidari et al., 2013). By contrast, clear differences were observed in phytochrome expression. In particular, PHYTOCHROME A (PHYA) transcripts were significantly less abundant in both pp2a-b′γ genotypes than in Col-0, with no difference between Col-0 and cat2 (Fig. 8b).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

A previous report on the pp2a-b′γ single mutant provided the first evidence that PP2A-B′γ is an actor in the control of defense responses in Arabidopsis (Trotta et al., 2011a). The present study provides new information on the role of this gene in determining day length-dependent responses to intracellular oxidative stress.

PP2A-B′γ is a key player in the negative control of day length-dependent SA-linked responses triggered by intracellular oxidative stress

Day length is emerging as an important control over responses of Arabidopsis to oxidative stress. In the cat2 single mutant grown at moderate light, lesions and associated pathogenesis responses are observed in LD but not SD (Queval et al., 2007; Chaouch et al., 2010). The LD-dependent responses are absent in cat2 sid2 and less apparent in cat2 npr1 (Chaouch et al., 2010; Han et al., 2013a). LD conditions also promote lesion formation in mutants for other genes implicated in cell death pathways, such as lesions simulating disease resistance1 (lsd1) and myo-inositol phosphate synthase 1 (mips1) (Dietrich et al., 1994; Meng et al., 2009), although whether these responses are associated with intracellular oxidative stress remains unclear. The notion that day length context influences responses to oxidative stress (rather than just oxidative stress intensity) is supported by studies of plants exposed for equal times to ozone in different day length regimes (Vollsnes et al., 2009; Dghim et al., 2013).

The present data clearly establish PP2A-B′’γ as a gene involved in the suppression of oxidative stress-driven lesions and related pathogenesis responses in SD. When introduced into the cat2 background, the pp2a-b′γ mutation triggered hallmarks of the SA signaling pathway that were otherwise not observed. As well as lesion spread and SA itself, markers included the accumulation of camalexin and PR1 transcripts. Activation of the pathway was also observed at the proteomic level. Although PR2 and PR5 phosphoproteins were not detected in the wild-type, both accumulated in the double mutant. Similar to cat2 growing in LD, the activation of lesions in cat2 pp2a-b′γ is dependent on the isochorismate pathway of SA synthesis, as lesions were absent in the triple cat2 pp2a-b′γ sid2 mutant.

Although the loss of CAT2 function is not sufficient to activate pathogenesis responses in SD, some responses were observed in the pp2a-b′γ single mutant in this condition. These included some accumulation of SA, although camalexin and PR1 expression remained at or close to wild-type levels. Although always less marked than in the double mutant, these responses in pp2a-b′γ were more pronounced in LD, conditions in which slight lesion formation was observed (Fig. 1), again underscoring the promotion of defense responses by long photoperiods. The activation of biotic stress defense responses in the pp2a-b′γ single mutant is in good agreement with the previous report of Trotta et al. (2011a), although some differences in the specific features of the responses were observed. Given the strong conditionality of plant responses to stress, differences could reflect the conditions used for the two studies. Phenotypes in the pp2a-b′γ single mutant are very sensitive to growth conditions, with lesions being less apparent as humidity or irradiance increases (A. Trotta & S. Kangasjärvi, unpublished). In the present study, both of these factors were higher than in that of Trotta et al. (2011a), possibly explaining why fewer lesions were observed in pp2a-b′γ. Despite their absence in both single mutants in SD in the growth conditions used in the present study, SA-dependent lesions were clearly apparent in cat2 pp2a-b′γ, underscoring the interaction between the two mutations.

A key role for PP2A-B′γ in regulating protein phosphorylation during oxidative stress

A number of phosphopeptides were differentially detectable when PP2A-B′γ function was lost in an oxidative stress context. Further studies will be required to establish whether these are substrates of PP2A oligomers containing this subunit, but three criteria can be used to identify the most interesting candidates: (1) the specificity for phosphoserine and phosphothreonine residues; (2) localization in the same compartments as PP2A-B′γ (cytosol, nucleus, plasmalemma; Table S4); and (3) increased phosphorylation status when PP2A-B′γ function is lost.

Based on these criteria, the best candidates to be direct targets during the oxidative stress response are SAHH, EF1α, HOL1, GSTF2, ACO2 and GLN1;1. The latter has previously been reported to be phosphorylated by calcium-dependent protein kinase (CDPK)-related kinase 3 (CRK3; Li et al., 2006), and so its phosphorylation status may be influenced by the opposing actions of this kinase and PP2A-B′γ-dependent phosphatase activity. The roles of PP2A-B′γ may not be restricted to PP2A-dependent dephosphorylation, but could also involve inhibition of the activity of a cytosolic calcium-dependent kinase (CPK1) through physical interactions (G. Konert et al., unpublished).

Whether or not these proteins are targets of PP2A-B′γ-dependent activities, our data provide clear evidence that this subunit is required to control the abundance or phosphorylation status of proteins during intracellular oxidative stress. Affected proteins include recognized players in redox homeostasis and associated signaling (HSP, GSTs, ACO2, CSD2, MDAR2, PR2, PR5). Indeed, GSTF2 has been described as a camalexin-binding protein (Dixon et al., 2011), which is in accordance with the strong accumulation of this phytoalexin in cat2 pp2a-b′γ (Fig. 5). Further, PDI2, CRT1 and CRT2 all reside in the lumen of the endoplasmic reticulum (ER) and are connected to the unfolded protein response during ER stress that is implicated in pathogenesis responses (Trotta et al., 2011b). Thus, PP2A-B′γ seems to be an integral part of the signaling hub that controls responses to oxidative stress at the proteomic level.

PP2A-B′γ as a central modulator of metabolic responses to oxidative stress

The pp2a-b′γ mutation not only allowed intracellular oxidative stress in SD to induce the accumulation of defense-related secondary metabolites, it also had a strong impact on the responses of primary metabolism. Intriguingly, SGAT was one of the proteins that showed opposite changes in abundance to the well-known SA-dependent PR2 and PR5 proteins (Table 2). This enzyme is one of at least two that act immediately downstream of H2O2-producing glycolate oxidase to convert glyoxylate to glycine, and has been implicated previously in pathogenesis responses (Taler et al., 2004). Our observations may suggest some co-regulation of the SGAT and CAT2 proteins. Although two independent microarray analyses show that SGAT transcripts are not significantly different in Col-0 and cat2 (Mhamdi et al., 2010c; Queval et al., 2012), there is some evidence that peroxisomal enzymes involved in photorespiration can associate into complexes (Heupel et al., 1991; Heupel & Heldt, 1994). The increased Ser in cat2 pp2a-b′γ may be a result of decreased SGAT abundance. Interestingly, however, Ser accumulation was less marked in the cat2 single mutant (Fig. S4). As well as its stimulation by the pp2a-b′γ mutation, cat2-triggered Ser accumulation is largely blocked by the sid2 mutation (Chaouch et al., 2010). This suggests that any physical interaction may be conditional, possibly linked to SA signaling. If so, this could be relevant to the described roles of peroxisomal H2O2 and SGAT activity in pathogenesis responses (Taler et al., 2004; Lipka et al., 2005; Rojas et al., 2012).

As well as Ser, many other amino acids are among the metabolites that are induced by cat2 in LD in an SA-dependent manner (Chaouch et al., 2010, 2012). Although much less or not accumulated in cat2 in SD, several of these amino acids were strongly induced in the double mutant (Fig. 4; Table 1; Fig. S4). The accumulation of amino acids was correlated with the induction of GLN1;1, a cytosolic glutamine synthetase (GS1), at both protein and transcript levels (Table 2; Fig. 8). Similar to the accumulation of the GLN1;1 protein in cat2 pp2a-b′γ, GS1 protein was up-regulated in tomato plants infected with Pseudomonas, whereas the chloroplastic GS2 was down-regulated (Pérez-Garcia et al., 1995). Opposing changes in the abundance of GS2 and GLN1;1 have also been observed previously in the pp2a-b′γ single mutant (Trotta et al., 2011a). As GS2 is the photorespiratory isoform, it is linked to SGAT and CAT2 functions. By contrast, GS1 is associated with nitrogen remobilization, for example, during senescence (Pérez-Garcia et al., 1995). One interpretation is that, in oxidative stress conditions, loss of PP2A-B′γ function down-regulates nitrogen cycling through the photorespiratory pathway in favor of the production of amino acids to support defense processes. This may occur to provide respiratory substrates for defense (Bolton, 2009), although other functions are possible. An amino acid transporter that may use glutamine as its major substrate has been implicated in the regulation of SA-dependent pathogenesis responses (Liu et al., 2010).

Mutations leading to the loss of regulation of enzymes involved in the synthesis of the aspartate-derived amino acids trigger changes in resistance to specific pathogens (Stuttmann et al., 2011). Alongside the activation of PR responses, isoleucine and Thr were both strongly accumulated in cat2 pp2a-b′γ, although two other aspartate-derived amino acids (homoserine and Met) were less affected. These last two compounds are involved in the Met cycle, a key source of methyl donors to various acceptors. Following methyl donation by S-adenosylmethionine (SAM), free homocysteine is regenerated by SAHH, proteins that were up-regulated in the double mutant (Table 2). Among many possible roles, methylation could be required for changes in secondary metabolism triggered by oxidative stress. Together with SAHH and PR proteins, cat2 pp2a-b′γ accumulated HOL1, which methylates the thiocyanate arising from the in vivo degradation of indolic glucosinolates (Nagatoshi & Nakamura, 2009). Methylation is also required in the synthesis of the coumarin, scopoletin, which accumulates in response to oxidative and biotic stress (Chong et al., 2002; Fig. 1). As well as being used in methylation reactions, SAM can be withdrawn from the cycle and used for the synthesis of ethylene, with the first and limiting reaction catalyzed by ACC synthase (ACS). A study of the roots curl in naphthylphthalamic acid1 (rcn1) mutant, which is deficient in a PP2A-A subunit, showed that two ACS isoforms were under differential control by PP2A-dependent processes (Skottke et al., 2011), whereas we found significant up-regulation of ACO2 in cat2 pp2a-b′γ, allowing phosphorylation sites to be detected in this polypeptide. Thus, it seems likely that PP2A regulates both enzymatic steps required to convert SAM to ethylene.

Relationship of PP2A-B′γ function to intracellular oxidative stress

Growing evidence indicates that the outcome of oxidative stress is conditioned by other factors, and not simply related to oxidative stress intensity. The finding that the pp2a-b′γ mutation allows cat2-triggered pathogenesis responses to be produced in SD implicates PP2A-B′γ as an influential actor in responses to intracellular oxidative stress.

Key features of intracellular oxidative stress in cat2 are the perturbation of intracellular thiol-disulfide status and the induction of a wide range of antioxidative genes (Mhamdi et al., 2010c; Queval et al., 2012), effects that are less evident in pp2a-b′γ (Fig. 3; Trotta et al., 2011a). In contrast with pp2a-b′γ (Trotta et al., 2011a) and cat2 pp2a-b′γ (this study), increases in DAB staining cannot be detected in cat2 growing in our conditions. There are thus clear differences in the redox effects driven by the two mutations. Further, their association in the double mutant produces synergistic or qualitative effects on defense responses in SD, that is, their combination either produces a greater than additive effect (SA and camalexin contents) or triggers responses that are undetectable or close to negligible in the single mutants (lesions, PR1 expression, several metabolites, proteomic profiles).

A key factor could be the interplay between ROS produced intracellularly and at the plasmalemma/apoplast. The activation of defense responses and the associated metabolic changes in cat2 in LD are influenced by AtRboh function, pointing to interactions between oxidative stress inside the cell and ROS produced at the cell surface (Chaouch et al., 2012). A primary role of PP2A-B′γ may be to restrict the activation of ROS production at the plasmalemma, explaining why the pp2a-b′γ mutant shows enhanced DAB staining (Trotta et al., 2011a), which mainly detects extracellular ROS. The appearance of enhanced DAB staining in cat2 pp2a-b′γ may suggest that the loss of PP2A-B′γ function weakens the repression of extracellular ROS production. This could cause increased sensitivity to cell-to-cell transmission of signals derived from perturbed intracellular redox status, triggering pathogenesis responses even in conditions in which they are not usually observed, and activating associated metabolic responses, such as nitrogen remobilization.

In conclusion, our study reveals that the repression of defense responses in SD is under genetic control through pathways linked to PP2A-B′γ function. Although further investigation is required to establish exactly how this is coupled to day length-dependent signaling, SA-dependent pathways are known to be modulated by photoreceptor functions (Genoud et al., 2002; Martínez et al., 2004; Griebel & Zeier, 2008). Decreased PHYA expression in the pp2a-b′γ genotypes (Fig. 8) suggests that photoreceptors may be involved in the regulatory nexus that links oxidative stress, day length, PP2A-B′γ and pathogenesis responses.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank the Salk Institute Genomic Analysis Laboratory (La Jolla, CA, USA) for providing the sequence-indexed Arabidopsis T-DNA insertion mutants and the Nottingham Arabidopsis Stock Centre (Nottingham, UK) for the supply of seed stocks. We are grateful to Patrick Saindrenan (IBP, Orsay, France) for the provision of HPLC facilities and the Turku Proteomics Facility (Turku, Finland) for excellent technical assistance. This work was supported by the European Union Initial Training Network ‘COSI’ and Academy of Finland projects 263772, 218157, 259888 and 130595.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office

FilenameFormatSizeDescription
nph12622-sup-0001-FigS1-S6.pptapplication/mspowerpoint1255K

Fig. S1 Production and genotyping of mutant lines.

Fig. S2 Ascorbate and glutathione contents in plants grown in long days (LD).

Fig. S3 Phenotypes and glutathione contents in mutants grown at low light.

Fig. S4 Amino acid contents in the cat2 pp2a-b′γ double mutant.

Fig. S5 Pattern of phosphoproteins in total soluble and membrane fractions isolated from wild-type, cat2, pp2a-b′γ and cat2 pp2a-b′γ leaves.

Fig. S6 Analysis of flowering time in Col-0 and mutant lines.

nph12622-sup-0002-TableS1.docxWord document17KTable S1 Primer sequences used in this study
nph12622-sup-0003-TableS2.xlsapplication/msexcel100KTable S2 Full list of metabolites detected by gas chromatography-time of flight-mass spectrometry (GC-TOF-MS)
nph12622-sup-0004-TableS3.xlsxapplication/msexcel20KTable S3 Ion scores, masses observed and expectation values for the identified phosphopeptides presented in Table 2
nph12622-sup-0005-TableS4.docxWord document35KTable S4 Likely subcellular localization of proteins modified in abundance or phosphorylation status in cat2 pp2-a-b′γ