Molecular Plant Advance Access originally published online on December 9, 2008
Molecular Plant 2009 2(2):357-367; doi:10.1093/mp/ssn084
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Redox-Dependent Regulation of the Stress-Induced Zinc-Finger Protein SAP12 in Arabidopsis thaliana
Biochemistry and Physiology of Plants, Bielefeld University, 33501 Bielefeld, Germany
1 To whom correspondence should be addressed. E-mail karl-josef.dietz{at}uni-bielefeld.de, fax +49 521 106 6039.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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The stress-associated protein SAP12 belongs to the stress-associated protein (SAP) family with 14 members in Arabidopsis thaliana. SAP12 contains two AN1 zinc fingers and was identified in diagonal 2D redox SDS–PAGE as a protein undergoing major redox-dependent conformational changes. Its transcript was strongly induced under cold and salt stress in a time-dependent manner similar to SAP10, with high levels after 6 h and decreasing levels after 24 and 48 h. The transcript regulation resembled those of the stress marker peroxiredoxin PrxIID at 24 and 48 h. Recombinant SAP12 protein showed redox-dependent changes in quaternary structure as visualized by altered electrophoretic mobility in non-reducing SDS polyacrylamide gel electrophoresis. The oxidized oligomer was reduced by high dithiothreitol concentrations, and also by E. coli thioredoxin TrxA with low dithiothreitol (DTT) concentrations or NADPH plus NADPH-dependent thioredoxin reductase. From Western blots, the SAP12 protein amount was estimated to be in the range of 0.5 ng µg–1 leaf protein. SAP12 protein decreased under salt and cold stress. These data suggest a redox state-linked function of SAP12 in plant cells particularly under cold and salt stress.
Key Words: abiotic/environmental stress • cold acclimation • cell signaling • gene expression • Arabidopsis • A20 and AN1 Zinc finger domains • redox regulation • stress associated protein • thioredoxin
Received for publication October 1, 2008. Accepted for publication November 4, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plants activate tolerance mechanisms upon demand within their given genetic plasticity. Sensitive sensing of deteriorating environmental conditions precedes the activation of the acclimation response to suboptimum conditions. Plants maintain a network of sensory mechanisms, signaling pathways and response systems to cope with these environmental challenges (Shinozaki et al., 2003; Fujita et al., 2006). The identification of components and the characterization of mechanisms of the response network are the subject of intensive research, albeit our understanding is only slowly emerging. Up-regulation of abscisic acid and ethylene levels mediates diverse stress acclimation responses (van Loon et al., 2006; Jiang and Hartung, 2008). Changes in membrane ion conductance and cytoplasmic calcium activities, protein kinase cascades, and various transcription factors have been identified as signaling components. Environmentally induced redox imbalances in cytoplasmic compartments have also emerged as major players in eliciting molecular responses of plants (Noctor, 2006; Dietz, 2008). Cellular redox properties change with varying intensity and are widely involved in modulating or triggering hardening and modifying developmental processes, mediated, for example, by thiol-/disulfide transition, glutathionylation, S-nitrosylation and nitration (Dalle-Donne et al., 2007). Redox proteomics approaches have revealed many potential targets of redox regulation that could be assigned to diverse functional categories (Ströher and Dietz, 2006; Buchanan and Balmer, 2005).
Recent work has shown that members of the A20/AN1 zinc-finger polypeptides are critically involved in stress tolerance of higher plants. Proteins with A20/AN1 zinc fingers have been identified in all eukaryotes and modulate cell functions in animals as diverse as immune response and muscle cell development (Huang et al., 2004; Hishaya et al., 2006). From a rice root cDNA library, Mukhopadhyay et al. (2004) isolated OsSAP1 (OSISAP1 for Oryza sativa Indica SAP1) and demonstrated high expression in roots and prepollination stage spikelets. SAP is an acronym for stress-associated protein. OsSAP1 transcript rapidly responds to cold, salt, heavy metals, mechanical wounding, and also to abscisic acid administration. Its heterologous expression in tobacco conferred dehydration, salt, and cold stress tolerance, at least in the seedling stage (Mukhopadhyay et al., 2004). OsSAP1 is a small 17.4-kDa protein of 164 amino acids with an N-terminal A20 domain characterized by a stretch of four Cys residues with variable intervening sequences and a C-terminal AN1 Zn-finger domain fitting to the consensus sequence Cx2–4Cx9–12Cx2Cx4Cx2Hx5HxC (x: any amino acid). The A20 domain is involved in dimerization reactions of animal homologues (de Valck et al., 1996). From their data, Mukhopadhyay et al. (2004) suggested that OsSAP1 might function in early stress response.
A genome-wide analysis of rice and Arabidopsis has identified 18 and 14 potential genes encoding SAP-like proteins, respectively (Vij and Tyagi, 2006). The SAP proteins can be grouped according to their domain structure. Ten genes (sap1–10) code for proteins with one A20 and one AN1 domain, each sap11 and 13 code for proteins with two AN1-domains and two and one A20 domain, respectively. SAP12 contains two AN1 zinc fingers, while SAP14 only contains one. Transcript profiling indicates a strong response to abiotic stress at least of a subgroup of the SAP family, suggesting a role of members of this family in stress acclimation. Our interest in SAP12 was based on its initial identification as a polypeptide subjected to significant redox-dependent conformational changes (Ströher and Dietz, 2008). In this study, 49 proteins belonging to the chloroplast proteome were identified.
Zinc-finger proteins have extraordinarily diverse functions, as they are not only involved in DNA/RNA recognition, but also enable binding to proteins or lipids (Laity et al., 2001). For the A20 and the AN1 zinc-finger domain of human Znf216, proteineous interaction partners have been already identified (Huang et al., 2004). Proteins with cysteine–zinc clusters often function as redox sensors, albeit Zn always remains in the Zn2+ state. Consequently, the redox targets are thiols in the Zn/S-coordination centre. Binding and release of the coordinated Zn atoms may be triggered by changing redox states of the thiols (Maret, 2006).
In search of signaling elements that might be involved in redox signal transmission and regulation, this work concentrated on the zinc-finger domain protein SAP12 due to its conformational switch in response to a changing redox environment. The aim was to establish a framework of its potential functional context by analyzing, for example, its transcript regulation relative to other family members and by determining the recombinant protein characteristics particularly with respect to the redox potential, the oligomerization state and the potential regeneration of reduced SAP12 by thioredoxin.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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A proteomic approach using diagonal 2D redox SDS–PAGE led to the identification of 74 proteins subjected to major redox-regulated conformational changes (Ströher and Dietz, 2008). Besides the 49 proteins belonging to the plastidic proteome, other proteins were identified but excluded from the above-mentioned study to focus only on the chloroplast redox proteome. The stress-associated protein SAP12 was one of those excluded proteins. SAP12 belongs to the family of A20/AN1 zinc-finger-containing stress-associated proteins (SAP) with 14 members in Arabidopsis thaliana (Vij and Tyagi, 2006). SAPs in general play a crucial role in response to abiotic stresses, as concluded from expression analysis in vivo and in silico (Vij and Tyagi, 2006). Indeed, transgenic plants in rice and tobacco are characterized by an enhanced tolerance to different abiotic stress treatments compared to the wild-type (Kanneganti and Gupta, 2008).
Bioinformatic Analysis
A phylogenetic tree was constructed with the members of the SAP family that can be assigned to different subgroups, depending on the organization of their domains (Figure 1A). SAP1–SAP10 have both the A20 and the AN1 zinc-finger domains clustered together, while those with two AN1 zinc-finger domains (SAP11–SAP13) or only one AN1 zinc-finger domain also clustered together. The analysis of the Affimetrix GeneChip data performed with genevestigator (https://iii.genevestigator.ethz.ch, Hruz et al., 2008) revealed slightly fluctuating transcript levels of the SAP family members during development from the seedling to the fully developed plant with mature fruits (Figure 1B). Compared to the other members, SAP2 has a higher transcript level throughout all analyzed stages, especially in the very early and late development. Application of various abiotic stresses such as cold, drought, genotoxic, osmotic, and oxidative effectors, salt and wounding for 6 h using hydroponics cultures (NascArrayReference No. 138), leads to a strong up-regulation of the transcript levels of the members of the SAP family. As the magnitude of this reaction is stronger in green tissues compared to roots, the present study focused on whole rosettes for the subsequent analyses. The following members of the SAP family were selected for further analysis, namely SAP2 as the member with the highest transcript abundance, SAP6, SAP9, and SAP10 due to the high up-regulation of their transcript amount in response to abiotic stresses, and SAP11 to SAP14 as a subgroup containing SAP12.
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Transcript Abundance
Transcript abundance of the selected SAP family members was assessed in response to different abiotic stresses by RT–PCR. To this end, 4–5-week-old plants grown on soil were subjected to salt (150 and 300 mM NaCl), hyperosmosis (15% (w/v) PEG-6000 with tentatively the same osmolarity as 150 mM NaCl) and cold (4°C) stress for 6, 24, and 48 h. In addition to transcript amounts of SAP2, SAP6, SAP9, SAP10, SAP11, SAP12, SAP13, and SAP14, those of PrxIIC and PrxIID were analyzed as well. Both were used as reference markers, as it was shown before that especially the mRNA level of PrxIIC is strongly up-regulated upon various stress applications (Horling et al., 2003). The plants showed a moderate stress phenotype at least after 48 h of stress treatment (Supplemental Figure 1). The transcript levels of the selected SAP family members particularly strongly responded to stress treatment. Especially SAP10 and SAP12 were already manifold up-regulated compared to the control treatment after 6 h, whereas SAP14 was undetectable at all, even when amplified with very high cycle numbers (Figure 2A). Compared to PrxIID, the mRNA level of PrxIIC was increased especially under high salt and cold treatment. After 24 h of stress treatment, the up-regulation of SAP10 and SAP12 was still obvious but decreased compared to 6 h (Figure 2B). The transcript amount of SAP2 remained the same under the different stress treatments during the whole experimental period, whereas SAP6, SAP9, SAP11, and SAP13 showed slight up-regulation, with the exception of cold stress treatment. The transcript amount of SAP14 first became detectable after 24 h in plants treated with 300 mM NaCl, 15% (w/v) PEG-6000 and cold (Figure 2B). The mRNA level of PrxIIC was still strongly increased compared to the control and for PrxIID up-regulation for high salt and cold stress was observed (Figure 2B). Compared to 24 h, the mRNA level of SAP6 at 48 h was further up-regulated under all abiotic stresses tested, whereas the transcript amount of SAP9 only remained up-regulated under high salt. The mRNA amount of SAP10 and SAP12 stayed high under high salt, decreased upon cold stress treatment, but was still up-regulated compared to the corresponding control (Figure 2C). The expression patterns of SAP11 and SAP13 were nearly preserved between 24 and 48 h. The regulation of the SAP12 transcript abundance under cold stress for the three analyzed time points was confirmed with quantitative real-time PCR (qRT–PCR) analysis. The strongest up-regulation of the SAP12 transcript amount was observed already 6 h after starting the cold treatment, where the transcript was nearly 200 times the control. It was slightly decreased to 140 times at 24 h and to 21 times at 48 h (Figure 2D). The quantitative RT–PCR and qPCR essentially showed the same transcript regulation of SAP12.
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Redox Regulation of SAP12 Protein
SAP12 was identified on the diagonal 2D redox SDS–PAGE as a protein undergoing major changes in electrophoretic mobility in dependence of its redox state. The amino acid cysteine is by far the most redox active proteineous amino acid (Davies, 2005) and oxidation to disulfide bridges has a large impact on the conformation of a given protein; therefore, the amino acid sequence of SAP12 was analyzed for the presence of (conserved) cysteines. In addition to the 12 cysteinyl residues involved in the coordination of the zinc within the zinc-finger structure, four additional cysteines were identified. Protein sequence alignment with SAP11–14 as a subgroup of the SAP family revealed one highly conserved cysteine (in SAP11–SAP14), two cysteines conserved in SAP11–13 and two additional conserved cysteines in SAP11 and SAP13 (Figure 3). The fourth cysteine of SAP12, whose redox-dependent regulation was the focus of this study, was not present at the same amino acid sequence position in any other SAP protein of this subfamily. Therefore, the above-mentioned three highly conserved cysteines appeared of higher interest in the context of contribution to the redox regulation of the protein, either in combination or independent of those cysteines that are responsible for the coordination of the zinc ions.
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For analysis of the redox-dependent behaviour of the SAP12, the full-length protein was heterologously expressed in E. coli and purified to homogeneity. In a first experiment contrasting oxidizing and reducing conditions were adjusted using different concentrations of the oxidant hydrogen peroxide (H2O2) and dithiothreitol (DTT) (Figure 4A). Different forms of the protein were detectable in non-reducing SDS–PAGE, namely a monomeric form at 24.6 kDa, a dimeric form at 55 kDa and several oligomeric forms. With increasing DTT concentrations coinciding with increased reducing power, the monomeric form became detectable, whereas, under oxidizing conditions, no signal was obtained, indicating the formation of high-molecular-mass aggregates. To confirm redox reversibility, the oxidized sample was re-reduced with high DTT concentration and the monomeric form of SAP12 could be visualized as a predominant form in the gel again (Figure 4B). For thiol–disulfide transitions, the redox midpoint potential Em° is a critical parameter. Em° in a strict sense characterizes the redox potential at which half of the cysteines are reduced whereas the other half are oxidized (Dutton, 1978). Here, it was determined for SAP12 using the mobility in a non-reducing SDS–PAGE as read out and adjusting the redox environment according to the method of Hirasawa and coworkers (1999). The transition by 50% from the monomeric form to the higher molecular forms with intermolecular disulfide bridges was realized at about –320 mV, as determined by densitometric analysis (Figure 5).
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To mimic the in vivo conditions, E. coli thioredoxinA (TrxA) instead of DTT was employed as electron donor for the regeneration of oxidized SAP12. Equimolar amounts were incubated in the presence of various DTT concentrations. Separation via SDS–PAGE and detection via Western blot analysis revealed that, indeed, TrxA promotes the regeneration of oxidized SAP12 already at 1 mM DTT (Figure 6A, bottom), compared to 5 mM DTT in the absence of TrxA (Figure 6A, top). The complete Trx regeneration system consisting of TrxA, thioredoxin reductase (TR), and NADPH was employed in a second experiment (Figure 6B). Again, the re-reduction of oxidized SAP12 could be detected using Western blot analysis. 1 mM NADPH was sufficient in the presence of the Trx regeneration system to reduce the dimeric form of SAP12 to the monomer lacking intermolecular disulfide bridges (Figure 6B).
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Quantification of SAP12 Amount in the Plant Tissue Using Specific Antibody
To study the SAP12 protein amount in the plant cell, a specific antibody was raised. The possibility of cross-reaction with other SAPs was tested with SAP2, which showed the highest transcript abundance in planta (Figure 1B). To this end, highly purified recombinant SAP2 was loaded in parallel to study the specificity of the antibody. SAP12 at 2 ng per lane was enough for detection with the specific antibody, whereas up to 10 ng of SAP2 gave no signal (Figure 7A). For the quantification of the SAP12 amount in whole rosette extracts, increasing concentrations of both highly purified SAP12 and leaf protein extract were employed. Signal density determination and comparison between SAP12 and leaf extract after Western blot analysis revealed about 0.5 ng SAP12 per µg leaf protein (Figure 7B). The SAP12 protein amounts responded to stress treatments as indicated by Western blot analysis of whole rosette protein extracts prepared after the different stress treatments in a time-dependent manner. After 48 h of stress treatment, 300 mM NaCl and 4°C, the protein amount decreased significantly (Figure 7C).
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Most plants experience deviations from optimum growth conditions during their lifecycle. If exceeding a basic level, such deviations are collectively called stress. Most stresses elicit deviation from a cellular redox norm that has to be readjusted. In light of the multiplicity of encountered stress factors and the significant costs of maintaining tolerance mechanisms, plants do not sustain stress tolerance mechanisms at a high level constitutively. This would be possible only with the trade-off of decreased fitness in terms of biomass production and diaspore generation (Haugen et al., 2008). Based on the previously published data on other SAP members and the results shown here, it is tempting to assume that the SAP protein family functions at the interface of redox signaling and stress acclimation. In vertebrates, proteins containing A20/AN1 domains are involved in immune response reactions (Huang et al., 2004). In 2006, a genome-wide study in Arabidopsis and rice revealed the presence of a large family of so-called stress-associated proteins (SAP) in plants (Vij and Tyagi, 2006). In recent studies, more details about single members of this family in rice were published. OsSAP1 (Mukhopadhyay et al., 2004), OsSAP8 (Kanneganti and Gupta, 2008), and Zfp177 (Huang et al., 2008) have been analyzed in some detail. All the studies focused on transcript analysis under different abiotic stresses and analysis of transgenic plants overexpressing the particular protein. Biochemical data are missing so far and are needed for assigning a function in the plant cell. This study provides new insight into the functional context of SAP12 under certain stress conditions in A. thaliana. To this end, a combination of transcript analysis, biochemical data, and bioinformatic predictions was employed.
Transcript Abundance in Response to Abiotic Stresses
Based on the transcript amount profiles of the SAP family members as analyzed by the genevestigator tool, eight SAP genes were selected and further investigated on the mRNA level in response to different abiotic stress treatment. Members of the SAP family in rice are known to respond fast to diverse abiotic stresses (Vij and Tyagi, 2006; Jin et al., 2007; Kanneganti and Gupta, 2008). The authors hypothesized that the members of the protein family exert their function especially in early phases of the stress response (Kanneganti and Gupta, 2008). A more detailed analysis of the large dataset available for A. thaliana revealed a differential and thus more specific response of the SAP transcripts. For example, SAP2 showed a constitutively high expression under all investigated abiotic stresses—possibly implying a housekeeping function of this protein. Furthermore, this study presents transcript profiles for SAP11 and SAP14, previously not accessible, for example, through gene chip hybridization. Interestingly, SAP14 is not detectable under non-stressed conditions, but increased after 24 h of stress treatment, while SAP12 belongs to those proteins whose transcript amounts react immediately after stress initiation.
In agreement with the experimental data for rice (Vij and Tyagi, 2006), the transcript analysis in A. thaliana presented in this study showed that the response pattern is not linked to the domain organization. For example, SAP10, an A20/AN1 protein, and SAP12, an AN1/AN1 protein, revealed a highly similar transcript regulation pattern under the investigated stresses and on the investigated timescale (Figure 2). In a recent study, the evolutionary distances between the single members of the protein family were calculated (Huang et al., 2008). Especially, the regions between the domains were shown to be highly variable. The authors suggested that these intervening regions underlie a high evolutionary pressure and might be responsible for functional diversification of the single SAP protein (Huang et al., 2008). Sequence alignment of the subgroup with the two AN1 zinc fingers allowed us to identify three cysteinyl residues that are highly conserved. In a converse manner, the first subgroup with A20/AN1 domains does not contain such conserved additional cysteines (Supplemental Figure 2). It is frequently encountered that plant proteins have a higher number of conserved cysteines, which take part in redox reactions (Ruelland and Miginiac-Maslow, 1999). In this light, the presence of the additional highly conserved cysteinyl residues tentatively indicates the existence of redox-regulated functions and/or interactions of SAP12.
Redox Regulation of SAP12 Protein
The family of stress-associated proteins came into the focus of our research after identifying SAP12 in the diagonal 2D redox SDS–PAGE (Ströher and Dietz, 2008). The observed redox-dependent change in electrophoretic mobility hinted at a major conformational change due to redox reactions that could be due to either inter- or intramolecular disulfide bond formation. The occurrence of the shift with pure recombinant protein shows that no other protein is needed for intermolecular disulfide bridge generation, but instead proves the existence of homodimers and -oligomers. In the cellular context, the most often encountered redox-reactive amino acid is the cysteine (for review, see Jacob et al., 2006). Among the 16 cysteines of SAP12, 12 are assigned to coordinate the zinc ions of the two zinc fingers present in each AN1 domain and, of the remaining four cysteines, three are conserved among the subgroup as pointed out above (Figure 3).
In response to differentially adjusted redox milieus, recombinant SAP12 adopted different putatively disulfide-linked forms, separating as monomeric, dimeric, or high-molecular-weight forms in non-reducing SDS–PAGE (Figure 4). Oxidizing conditions are known to release the zinc ion from the zinc-finger structures in some proteins (Maret, 2006). To exclude conformational changes due to the disassembly of the zinc fingers, a zinc assay was performed. Zinc ions were released under strongly oxidizing conditions (data not shown). This suggests that the conformational changes in response to less drastically changing redox conditions (Figure 4) are mediated by redox reactions of the other cysteinyl residues located between the zinc-finger structures.
The midpoint potential of SAP12 was determined to be about –320 mV. Due to the absence of any clear signal peptide, TargetP (www.cbs.dtu.dk/services/TargetP/; Emanuelsson et al., 2007) and other bioinformatics tools predict that SAP12 is a cytosolic protein. Taken into consideration that the potential of the cytosol in A. thaliana epidermis cells is at about –320 mV, as demonstrated in a recent study employing reduction–oxidation-sensitive GFP (roGFP) (Schwarzländer et al., 2008), the conformational state of SAP12 may vary in tight linkage to changes in the cytosolic redox state, by shifts either to more reducing or to more oxidizing conditions. However, it should be noted that the redox state of the cellular Trx pool is not necessarily in equilibrium with the GSH pool sensed by roGFP.
An important feature of redox-dependent conformational changes is their reversibility, which enables a fast response to changing environmental conditions and makes thiol-disulfide transitions a powerful mechanism in cellular regulation (Ströher and Dietz, 2008). The thioredoxin regeneration system consisting of thioredoxin, thioredoxin reductase, and NADPH is the best studied system for supplying electrons to oxidized protein thiols (for review, see Meyer et al., 2008). The list of interaction partners has grown to more than 300 proteins and still has to be expanded (Buchanan and Balmer, 2005; Dietz, 2008). The data presented in this study reveal that SAP12 is an interaction partner and electron acceptor of the Trx system. The cytosol contains a set of thioredoxins of the Trx h type with apparently overlapping function (Gelhaye et al., 2004; Reichheld et al., 2008).
Function of SAP12 In Planta
Transcript analysis revealed a low level of expression of SAP12 mRNA under control conditions as, for example, compared to SAP2 or SAP6 (Figure 1B). The SAP12 protein in planta as estimated with a specific antibody raised in rabbit with highly purified recombinant SAP12 protein amounted to approximately 0.05% of the total leaf proteome. It appears to be higher than expected for a protein with catalytic function in regulation like protein kinases. Cross-reactivity of the antibody at least with SAP2 was excluded (Figure 7). The constantly high protein amount is in contrast to the strong induction of transcript abundance and might be due to higher turnover of the mature protein under abiotic stress conditions—a hypothesis that will have to be analyzed in more detail in future work. However, after 48 h of stress, the protein amount resembled the transcript pattern and was down-regulated (Figure 7). This supports the testable hypothesis that the function of SAP12 protein is important in the early phase of stress response. First initial experiments employing non-reducing SDS–PAGE with stressed plant material were performed, showing small shifts in the apparent molecular mass of SAP12 under salt and cold stress, possibly indicating the formation of intramolecular disulfide bonds. This result will be the topic of further work.
Functional assignment for the members of the A20/AN1 proteins was not possible due to the absence of the biochemical data until now. A20/AN1 zinc-finger protein interacts with protein (Matthews and Sunde, 2002), and, for human Znf216, it was shown that the AN1 domain interacts with an E3 ubiquitin ligase TRAF6 (Huang et al., 2004). Detailed analysis of the binding was not performed; therefore, no clear binding motif for the AN1 domain is known. Blast analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the Arabidopsis genome reveals a distant homolog COP1 with sequence identity of 40%. COP1 also encodes an E3 ubiquitin ligase, which is, in general, responsible for recognition and recruitment of substrates for modification with ubiquitin for subsequent degradation (for review, see Kerscher et al., 2006). Co-expression analysis over all 322 available ATH1 microarrays was performed using the tool ACT (Arabidopsis co-expression data mining tool; www.arabidopsis.leeds.ac.uk/act/index.php; Manfield et al., 2006) to get a first hint towards the regulatory context in the plant cell and possible interaction partners. Interestingly, the transcript of COP1 is negatively co-regulated with SAP12 (Figure 8A). COP1 is involved in the negative regulation of the process of photomorphogenesis in the absence of light (Yamamoto et al., 1998). A possible interaction between SAP12 and COP1 and the implications for the plant cell have to be further analyzed.
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Co-expression analysis also reveals the positive co-expression with Rap2.6, a transcription factor belonging to the ERF (ethylene response factor) subfamily B-4 of the overall superfamily of APETALA2(AP2)-ethylene-responsive element binding proteins (EREBP) domain-containing transcription factors (for review, see Nakano et al., 2006). Recently, Rap2.6 was identified as one of the 16 most responsive genes towards diverse abiotic stresses, the so-called ‘Multiple Stress Regulatory’ (MSTR) genes (Kant et al., 2008). Rap2.6 and Rap2.1 are involved in the cold response pathway in plants by mediating signals from the CBF (C-repeat-binding factor) regulon to adapt gene expression of further downstream elements (Fowler and Thomashow, 2002). In addition, other transcription factors of the ERF family of the AP2/EREBP superfamily ERF1 and ERF#011 were positively co-expressed with SAP12 (Figure 8A). At least for ERF#011, it is known to be involved in salt stress response (Dinneny et al., 2008). Target genes have not been identified so far, but the co-expression might indicate an interrelation with SAP12. Promotor analysis showed the presence of AP2/EREBP binding sites in the upstream region of SAP12 (Table 1). Therefore, it might be concluded that the transcript abundance of SAP12 might be regulated via different AP2–EREBP transcription factors, in dependence of the corresponding abiotic stress. These hypotheses are interesting topics for future investigations. Co-expression analysis over all available experiments and transcript analysis under abiotic stresses using the genevestigator tool also led to the identification of two cytosolic thioredoxins, H5 and H9 (Figure 8B), which might be responsible for the regeneration of oxidized SAP12.
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In conclusion, based on the overall midpoint redox potential and the reversibility of oxidation by the Trx system, it is tempting to speculate that SAP12 acts as a redox sensor, transmitting redox information to other components of the cell, most likely proteins that bind to SAP12. Two scenarios may be suggested, namely that binding of one particular redox conformation either acts as inhibitor or as activator of interacting proteins.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plant Material and Growth Conditions
A. thaliana ecotype Columbia was grown on soil under controlled conditions (10 h light at 100 µmol quanta m–2 s–1, 23°C and 14 h darkness at 18°C; 50% relative humidity). Four to 5-week-old plants were used for the analysis of the transcript amounts of selected transcripts. Four hours after onset of illumination, selected stress treatments were started. Cold stress was performed at 4°C, salt stress was applied by extensively watering the pots with 200 ml of 150 and 300 mM NaCl solution, respectively. Likewise, the osmotic treatment was performed with 15% (w/v) PEG-6000 corresponding to the osmolarity of the 150 mM NaCl solution. Complete rosettes of control and treated plants were harvested after 6, 24, and 48 h, immediately frozen in liquid nitrogen and stored at –80°C.
RNA Isolation, RT–PCR, and qRT–PCR
RNA isolation and the subsequent cDNA synthesis were performed according to Wormuth et al. (2006). Supplemental Table 1 lists the employed primer combinations.
Quantitative real-time PCR (qRT–PCR) was performed on the iCyclerTM Thermal Cycler (Bio-Rad, USA) with the iQTM SYBR Green Supermix (Bio-Rad, USA) according to the manufacturers’ instructions. Subsequent melting curve analysis confirmed the specificity of the employed primers (Supplemental Table 1). Agarose gel electrophoresis verified the expected fragment size. Data were normalized to actin and PCR efficiencies and threshold cycle values for each primer/template combination were used to calculate expression levels.
Cloning, Expression, and Purification of Recombinant SAP12 and Production of Specific Antibody for SAP12
The coding sequence of SAP12 was amplified using the primer combination given in Supplemental Table 1. After digestion with BamH1 and EcoR1, the amplified fragments were ligated into the modified 35S–YFP vector (Seefeldt et al., 2008). Protein was expressed and purified according to Ströher and Dietz (2008). Protein amounts were quantified using the Bio-Rad protein assay (Bio-Rad, Munich, Germany) according to the manufacturer's instructions.
Purified recombinant protein was employed to raise an antiserum against SAP12 in rabbit (Pineda, Berlin, Germany).
Redox Titration
Redox potentials (–350, –330, –310, –300, –290, –270, –250 mV) were calculated with the Nernst equation for a two-electron reaction and adjusted using defined ratios of dithiothreitol DTTred/DTTox with a total DTT concentration of 50 mM according to Hirasawa et al. (1999). All Em value calculations were based on a value of –330 mV for the Em of DTT at pH 7.0 (Hutchison and Ort, 1995). Reducing and oxidizing conditions were adjusted with DTTred or DTTox alone, respectively. Thiol reshuffling was prevented by alkylation with 100 mM iodoacetamide (IA) at room temperature in the dark for 30 min. Non-reducing SDS sample buffer was added and the protein samples were subjected to polyacrylamide gel electrophoresis.
Regeneration of Oxidized SAP12
(1) Regeneration with recombinant E. coli TrxA and DTT. Equimolar concentrations (3.4 µM) of heterologously expressed SAP12 and E. coli TrxA were incubated in 50 mM MES pH 7.0 in the presence of different concentrations of DTT (0, 0.1, 1, 5, and 50 mM) for 10 min at RT. SAP12 or TrxA alone served as control.
(2) Regeneration with recombinant E. coli TrxA, thioredoxin reductase (TR), and NADPH. Equimolar concentrations (3 µM) of SAP12, TrxA, and TR were incubated with different concentrations of NADPH (0, 0.1, 0.5, 1, and 5 mM) in 40 mM K–Pi pH 7.0 for 10 min at RT.
Free thiols were alkylated by addition of 100 mM IA and incubation at RT in the dark for 30 min. Non-reducing SDS sample buffer was added and the samples were subjected to electrophoresis.
Protein Extracts
Plant tissue from stressed plants was ground to fine powder in liquid nitrogen. Proteins were extracted in a buffer containing 500 mM Tris-HCl (pH 6.8). Protein concentrations were determined using the BioRad protein assay (Bio-Rad, Munich, Germany) according to the manufacturer's instructions. Reducing SDS sample buffer was added and the protein samples were heated to 95°C for 5 min prior to electrophoresis.
SDS–PAGE and Immunoblotting
Electrophoresis was performed on either a discontinuous gel consisting of a separating gel of 12% (w/v) acrylamide and a stacking gel of 6% (w/v) acrylamide or, if indicated, a continuous gradient gel ranging from 5 to 12% (w/v) acrylamide to enhance the separation of high-molecular-mass complexes. After completion of the separation, Western blot analysis was performed for the detection of SAP12 either with specific His-tag antibody or with specific anti-SAP12 antibody, depending on the specific application.
Phylogenetic Analysis
The A. thaliana SAP protein sequences were aligned using ClustalW version 2.0 with default options; only for the alignment of the subgroup SAP11 to SAP14 a gap extension value of 5 was chosen (www.ebi.ac.uk/Tools/clustalw/; Larkin et al., 2007). The alignments are visualized using JALVIEW version 2.3 (Clamp et al., 2004). For phylogenetic analysis of the SAP family, the alignment was imported into MEGA version 2.1 (Kumar et al., 2008) and the phylogenetic tree constructed using the neighbor-joining method with Poisson correction. Bootstrap analysis was performed with 1000 replicates.
Accession Numbers
Sequences used in this study can be identified in NCBI nucleotide or protein data libraries under the following accession numbers: SAP2 (AT1G51200), SAP6 (AT3G52800), SAP9 (AT4G22820), SAP10 (AT4G25380), SAP11 (AT2G41835), SAP12 (At3g28210), SAP13 (AT3G57480), SAP14 (AT5G48205), PrxIIC (At1g65970), PrxIID (At1g60740), Actin (At5g09810), Trxh1 (AT3G51030), Trxh3 (AT5G42980), Trxh4 (AT1G19730), Trxh5 (AT1G45145), Trxh6 (AT2G40790), Trxh9 (AT3G08710), TrxCxxS2 (AT1G11530), Picot1 (AT4G04950).
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This work was funded by the Deutsche Forschungsgemeinschaft DFG.
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This work was performed within the Research Centre FOR 804. No conflict of interest declared.
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