Molecular Plant Advance Access published online on November 17, 2008
Molecular Plant, doi:10.1093/mp/ssn070
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Photosynthetic Regulation of the Cyanobacterium Synechocystis sp. PCC 6803 Thioredoxin System and Functional Analysis of TrxB (Trx x) and TrxQ (Trx y) Thioredoxins
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Avda Américo Vespucio 49, 41092-Sevilla, Spain
1 To whom correspondence should be addressed. E-mail floren{at}us.es, fax +34-954460065, tel. +34-954489509.
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The expression of the genes encoding the ferredoxin–thioredoxin system including the ferredoxin–thioredoxin reductase (FTR) genes ftrC and ftrV and the four different thioredoxin genes trxA (m-type; slr0623), trxB (x-type; slr1139), trxC (sll1057) and trxQ (y-type; slr0233) of the cyanobacterium Synechocystis sp. PCC 6803 has been studied according to changes in the photosynthetic conditions. Experiments of light–dark transition indicate that the expression of all these genes except trxQ decreases in the dark in the absence of glucose in the growth medium. The use of two electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), reveals a differential effect on thioredoxin genes expression being trxC and trxQ almost unaffected, whereas trxA, trxB, and the ftr genes are down-regulated. In the presence of glucose, DCMU does not affect gene expression but DBMIB still does. Analysis of the single TrxB or TrxQ and the double TrxB TrxQ Synechocystis mutant strains reveal different functions for each of these thioredoxins under different growth conditions. Finally, a Synechocystis strain was generated containing a mutated version of TrxB (TrxBC34S), which was used to identify the potential in-vivo targets of this thioredoxin by a proteomic analysis.
Key Words: Cyanobacteria • photosynthetic electron transport • Synechocystis • oxidative stress • thioredoxin
Received for publication July 30, 2008. Accepted for publication October 2, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Thioredoxins are small proteins involved in disulphide/dithiol reactions and serve as redox carriers in a variety of physiological processes, including enzyme activity regulation, DNA metabolism, or acting as a reducing factor in several biosynthetic pathways (Holmgren, 2000). In general, all thioredoxins contain a cysteine disulphide active site, with the conserved amino acid motif Trp–Cys–Gly–Pro–Cys. However, new thioredoxins contain amino acid changes in this motif, but conserve both cysteines (Buchanan and Balmer, 2005; Holmgren, 2000). Thioredoxins are involved in light-dependent enzyme regulation of many oxygen-evolving photosynthetic organisms from cyanobacteria to higher plants, recently reviewed by Schurmann and Buchanan (2008). In its reduced form, thioredoxin recognizes and reduces specific disulphide bonds of several target proteins, such as NADP-malate dehydrogenase or fructose 1,6 bisphosphatase, to trigger a change in their catalytic activity (Buchanan et al., 1994; Lemaire et al., 2007; Schurmann and Buchanan, 2008).
In higher plants, thioredoxins belong classically to three different types, two chloroplastic (m and f-type) and one localized in the cytosol (h-type), but recently the universe of thioredoxin has been increased with the analysis of sequenced genomes. New thioredoxin types, such as mitochondrial thioredoxin (Trx o) and new chloroplastic thioredoxins (Trx x and Trx y) have been described (Buchanan and Balmer, 2005; Collin et al., 2003; Gelhaye et al., 2005; Lemaire et al., 2003, 2007; Meyer et al., 2005, 2008). Furthermore, the existence of several monocysteinic thioredoxins, with the active site sequence CGFS, has been also reported (Serrato et al., 2008). In cyanobacteria, the thioredoxin group is more complex than was previously considered, and, until the sequencing of the genome of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), only the thioredoxin with similarity to m-type thioredoxins was studied in cyanobacteria, with the exception of a unusual thioredoxin form in Anabaena sp. PCC 7120 (Gleason, 1992; Gleason and Holmgren, 1988; Muller and Buchanan, 1989; Navarro and Florencio, 1996). The complete sequence of the Synechocystis genome revealed three ORFs (sll1057, slr0233, and slr1139) that code for three thioredoxins apart from trxA (slr0623) gene encoding the m-type thioredoxin (Florencio et al., 2006; Hisabori et al., 2007).
To study the regulation of genes of the FTR–TRX system in a cyanobacterium, we have used Synechocystis, where gene expression of the entire genome following changes in different growth conditions has previously been analyzed, including high light intensity or the use of the photosynthetic electron transport inhibitors DCMU and DBMIB (Hihara et al., 2001, 2003). Detailed studies of the photosynthetic genes psbA, psbD, psaA, or psaB (Mohamed et al., 1993; Mohamed and Jansson, 1989; Smart and McIntosh, 1991), the rbcLS operon (Mohamed and Jansson, 1991), and the nitrogen-regulated glnA and glnB genes (Garcia-Dominguez and Florencio, 1997; Reyes and Florencio, 1995) as a function of light and/or electron transport have previously been reported. In addition, previous work showed that Synechocystis trxA transcript accumulation is similar under heterotrophic, photoheterotrophic, mixotrophic, and photoautotrophic growth conditions (Navarro et al., 2000).
In this study, we show that expression of the different thioredoxin genes responds to photosynthetic conditions in two manners; one group, which includes trxA and trxB and the FTR genes, exhibits photosynthetic dependence, whereas a second group that includes the trxQ and trxC genes displays a relatively independent expression of the photosynthetic growth conditions. Furthermore, we analyzed the role of Synechocystis TrxB and TrxQ corresponding to the higher plants thioredoxins Trx x and Trx y, respectively. TrxB mutant strain exhibits higher tolerance to diamide and is more sensitive to DTT and high light intensity, whereas TrxQ mutant is affected in cell growth in the presence of hydrogen peroxide. Finally, a site-directed mutant of TrxB, where the resolving cysteine was mutated to serine, was introduced in Synechocystis replacing endogenous TrxB; in this Synechocystis strain, we were able to trap different proteins that could be potential targets for this thioredoxin in vivo, according to our proteomic study.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Expression of the FTR–TRX System under Different Photosynthetic Conditions
In cyanobacteria, light exerts a fundamental role on gene expression. The effect of light and the photosynthetic electron transport (PET) through both photosystems has been analyzed by microarray analysis or by Northern blot of specific genes in Synechocystis (Hihara et al., 2001, 2003; Navarro et al., 2000). We have studied the expression of the FTR–TRX genes in the light or after transfer to darkness, in the presence or in the absence of glucose. As shown in Figure 1A, all the analyzed genes were expressed in the light but with the exception of trxQ transcription declined about 80–90% after transfer to darkness for 12 h (Figure 1B). The expression of all these genes in the presence of glucose (in the light or in the dark) in the growth medium decreased about 20–30% with respect to the levels detected in the light (Figure 1B).
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To analyze this effect in more detail, we proceeded to perform light–dark transitions over short time periods for studies of the gene expression of the FTR–TRX genes. To this end, Synechocystis cells were grown in the light (50 µE m–2 s–1) and thereafter transferred to darkness for a short time (4 h) and then back to light. As shown in Figure 2A, ftr genes as well as the trxA and trxB genes reduced their expression in the dark, whereas trxC and trxQ expression levels appeared less affected. However, re-illumination of the cultures activated the expression of all these genes, reaching levels previously observed in the light or even higher (Figure 2B). In order to determine if the rapid decay of trx and ftr transcripts, except trxC and trxQ transcripts, in the dark was due to different stability of the mRNA in light versus darkness, the half-life of different mRNA were determined under light and dark conditions after rifampicin addition. As shown in Supplemental Figure 1, the transcript stabilities were similar in light (t1/2 3 min) and in darkness (t1/2 3 min).
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The above results suggested that transcription of the FTR–TRX genes are regulated in some way via the PET between both photosystems. In order to obtain a more detailed analysis of this effect, we used two different photosynthetic electron inhibitors that act at different levels: DCMU, which blocks electron flow between PSII and the plastoquinone pool, and DBMIB that prevents the cyclic electron flow, impairing the oxidation of the plastoquinone pool by the cytochrome b6f complex. Both DCMU and DBMIB inhibit oxygen evolution at 10 µM in Synechocystis (Hihara et al., 2003; Reyes and Florencio, 1995). As shown in Figure 3A, the levels of the different transcripts decreased after 1 h of DCMU addition, except for trxC and trxQ, while, after 4 h of treatment, all transcript levels were reduced to about 20%, and only trxQ remained high (about 80% of the original level) (Figure 3B). When DBMIB was added to Synechocystis cells, both trxC and trxQ expression remained unaffected after 2 h of treatment. However, ftr and trxA and trxB transcripts rapidly decreased, to about 20% of the initial level within 1 h of DBMIB treatment (Figure 3A and 3B). Interestingly, when DBMIB was added to Synechocystis cells grown mixotrophically, the different transcript levels responded similarly to the same addition to photoautotrophic growing cells (Figure 4A), whereas the DCMU addition to mixotrophically grown cells did not promote any transcript change compared with the untreated control cells (Figure 4A), with the exception of trxA transcript, which increases up to 150% (Figure 4B). This indicates that, in the case of DCMU, the presence of glucose is sufficient to maintain the transcription of the different genes analyzed (Figure 4A and 4B).
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TrxB (x-Type) and TrxQ (y-Type) Thioredoxins Are Non-Essential for the Cyanobacterium Synechocystis under Normal Photoautotrophic Growth Conditions
To identify the specific functions of each thioredoxin, TrxA, TrxB, TrxC, and TrxQ, we attempted to generate mutants by disruption of the corresponding genes with an antibiotic resistance cassette.
It was not possible to obtain a strain with all the chromosomal copies of trxA mutated, as was previously reported (Navarro and Florencio, 1996). In the case of the trxC gene, a fully segregated mutant could not apparently be obtained, even after many rounds of segregation at increasing antibiotic concentration (data not shown), suggesting that, similar to TrxA, this thioredoxin is also essential under normal photoautotrophic growth conditions of Synechocystis.
However, fully segregated mutants of the thioredoxins TrxB and TrxQ were obtained. The trxB gene was disrupted by a streptomycin/spectinomycin cassette (Spr/Spr) in both orientations (+ and –) (Figure 5A). The resultant strains were named as STXB+/–. On the other hand, the trxQ sequence gene was disrupted by a kanamycin/spectinomycin cassette (Kmr) in both orientations (Figure 5A). The resultant strains were denoted as STXQ+/–. Similar results were obtained with both orientations for the two different thioredoxins. Thus, only data about the mutants with the positive orientation of the resistance cassette with respect to the thioredoxin gene are shown. A double mutant lacking trxB and trxQ genes was also generated by using both cassette resistance genes. This strain was named STXQB. The correct integration and segregation of each mutant were confirmed by Southern blot analysis (Figure 5B), with the wild-type (WT) strain being used as control. The trxB and/or trxQ insertions were fully segregated, indicating that TrxB and TrxQ are not essential under normal growth conditions.
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Characterization of STXB and STXQ Mutant Strains
We investigated the effect of trxB and/or trxQ disruption on cell viability. Under normal light conditions (50 µE m–2 s–1), the growth and chlorophyll levels of all thioredoxin mutants were the same as those of WT cells (Figure 6A). However, when cells were transferred to high light conditions (500 µE m–2 s–1), STXB mutant grew considerably slower than WT strain, displaying a growth rate similar to that observed under normal light conditions (Figure 6A), suggesting a possible role for TrxB thioredoxin in the adaptation to high light conditions. On the other hand, the STXQ strain was slightly less tolerant to hydrogen peroxide compared to the WT strain when cells were growing photoautotrophically (Figure 6B). Therefore, TrxQ may be involved in the oxidative stress response in the cyanobacterium Synechocystis, as suggested for thioredoxin y-type of higher plant (Collin et al., 2004; Lemaire et al., 2003).
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, 
) and STXB (
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) mutant cells under normal (open symbols) (50 µE m–2 s–1) and high light (closed symbols) (500 µE m–2 s–1) illumination conditions.Furthermore, the STXB and STXQ mutant strains were examined for tolerance to diamide or DTT. To this end, WT and mutant strains were spotted onto BG11C plates supplemented with diamide (100 and 250 µM). The WT and the STXQ strains were sensitive to diamide at 100 µM final concentration, STXQ being the most sensitive strain, whereas the STXB mutant exhibited a clear tolerance even at high concentration (250 µM) of diamide, suggesting that the absence of TrxB makes Synechocystis more tolerant to the presence of this oxidizing agent. The STXQB double mutant showed an intermediate phenotype between the STXB and STXQ strains, being almost similar to the WT strain (Figure 6C). Thus, these results suggest that other antioxidant systems are present in Synechocystis cells that may be induced or more active in the STXB mutant; one component of this system could be TrxQ thioredoxin.
When these strains were spotted onto BG11C plates containing different DTT concentrations (5 and 10 mM), the STXB strain clearly showed more sensitivity to DTT than WT or STXQ strains (Figure 6C). The double mutant (STXQB) displayed an intermediate behaviour compared to single mutants. As expected, SBC34S cells containing a non-functional TrxB behaved as STXB cells, namely resistance to diamide and sensitivity to DTT, especially at high DTT concentration (10 mM).
Thus, these results suggest that x-type thioredoxin TrxB might be an important partner in the reductive stress tolerance response in Synechocystis.
Construction of a Synechocystis Strain that Expresses a His-Tagged Cysteine-to-Serine TrxBC34S Mutant Protein
We have previously reported a detailed analysis of the in-vitro target proteome of the TrxA, TrxB, and TrxQ thioredoxins in the cyanobacterium Synechocystis (Lindahl and Florencio, 2003; Mata-Cabana et al., 2007; Perez-Perez et al., 2006). Here, we have developed a different approach to obtain the in-vivo target proteome for TrxB. To this end, a His-tagged form of the TrxB site-directed mutant TrxBC34S (Perez-Perez et al., 2006) was expressed in the STXB mutant strain, where the endogenous trxB gene was previously disrupted (Figure 5). The trxBC34S mutant gene was inserted into nrsD (slr0796), a non-essential gene in Synechocystis under normal growth conditions that belongs to the nickel resistance system (Lopez-Maury et al., 2002) (Figure 7A). The resulting strain was named SBC34S. The correct integration in the genome and the complete segregation of the mutation were analyzed by Southern blot. As shown in Figure 7B, the size of the resulting bands indicated that the TrxBC34S gene was inserted at the correct position.
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We next confirmed the expression of TrxBC34S in the SBC34S STXB strain by Western blot analysis using an antibody raised against Synechocystis TrxB (Florencio et al., 2006). TrxB and TrxBC34S were detected in WT and SBC34S cells, whereas no signal was found in the STXB mutant (Figure 7C). Furthermore, to investigate the correct expression of the 6xHis tag in the TrxBC34S protein, we performed Western blot analysis of WT, STXB, and SBC34S cells using an anti-6xHis antibody. A single band was detected exclusively in the SBC34S strain (Figure 7D). Therefore, these results demonstrated that the TrxB mutant version was properly expressed in SBC34S cells to a level that was slightly higher than the one observed in WT cells. Interestingly, under non-reducing conditions, several bands of higher molecular weight in addition to the TrxB monomer were recognized by the anti-TrxB antibody in total extracts from SBC34S cells but not in WT cell extracts (Figure 7C). Treatment with a reducing agent such as dithiothreitol (DTT) resulted in a decrease in the intensity of some of these bands together with the proportional increase in the intensity of the TrxB monomer (Figure 7C). Thus, the signals obtained under non-reducing conditions correspond either to complexes between TrxB and putative targets, or to oligomers of TrxB.
Identification of In-Vivo TrxB Targets
TrxB targets were purified from total extracts obtained from SBC34S cells by Nickel-affinity chromatography as described in the Methods section. Elution fractions were subjected to Western blot analysis using an anti-TrxB antibody to confirm the presence of the different TrxB-target complexes (Figure 8A). Those fractions in which the intensity of the putative TrxB targets was higher (F4 and F5) were combined and subjected to 2-D non-reducing/reducing gels. As previously described in the study in which the in-vitro targets of TrxB were identified (Perez-Perez et al., 2006), TrxBC34S showed a distinctive pattern of intense spots along the horizontal row that corresponded to different oligomeric forms of this thioredoxin due to the presence of an additional cysteine (Cys69) in this protein. Cys69 is also responsible for the fact that TrxBC34S does not produce distinct spots corresponding to its target proteins, but rather each of its targets gives rise to a train of spots with a streaky appearance (Figure 8B).
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The putative TrxB targets were identified by mass spectrometry as described in the Methods section. Table 1 summarizes the proteins that have been identified in at least three independent experiments. As a result of our in-vivo approach, we have identified 24 proteins as potential targets of TrxB (Table 1). Nine of these proteins have been previously identified as TrxA, TrxB, or TrxQ targets in vitro (Lindahl and Florencio, 2003; Perez-Perez et al., 2006). Some of these proteins, such as phosphoribulokinase (Balmer et al., 2003; Lemaire et al., 2004), glyceraldehyde-3-phosphate dehydrogenase (Balmer et al., 2003; Marchand et al., 2004; Wong et al., 2002; Yamazaki et al., 2004), and transketolase (Balmer et al., 2003; Marchand et al., 2004), have also been reported as thioredoxin targets in different photosynthetic organisms, validating our method.
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The highest MOWSE scores obtained by MASCOT for the targets are indicated in Table 1. The number of cysteine residues in each identified protein ranges from one to nine.
As a result of our previous in-vitro analysis of the target proteome of Synechocystis thioredoxins (Lindahl and Florencio, 2003; Perez-Perez et al., 2006), we identified 39 proteins as putative targets of TrxA (m-type), TrxB (x-type), and TrxQ (y-type). These proteins are involved in several cellular processes such as carbon dioxide fixation, nitrogen metabolism, light harvesting or oxidative stress response. In this in-vivo approach, we have found new proteins as TrxB targets but they belong to the same metabolic pathways. Proteins identified for the first time in this study include some ribosomal proteins from the large subunit, such as the L11 methyltransferase, L3, L24, and L28, as well as S2 and S10 from the small subunit. The target proteome of Synechocystis TrxB also includes proteins involved in nitrogen metabolism such as glutamine synthetase or cyanophycinase, or proteins with a role in sulphur metabolism such as cysteine synthase.
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Expression of the FTR–Trx Genes under Different Photosynthetic Conditions
Several studies have analyzed the Synechocystis whole-genome transcription profile in relation to the photosynthetic growth conditions, such as high light effects (Hihara et al., 2001; Huang et al., 2002), or light–dark transitions (Gill et al., 2002). High light treatment promotes the decrease in the levels of the transcripts of many genes related with the photosynthetic apparatus, especially PSI and phycobilisome genes, whereas PSII genes such as psbA2 and psbA3 increase their expression together with CO2 concentration mechanism and fixation (Hihara et al., 2001; Huang et al., 2002). Our previous results demonstrated that trxA also decreases after shifting to high light intensity (Navarro et al., 2000). In the light–dark transition analysis, about 783 genes respond positively to light, with three different time-course groups—one responds rapidly, a second group after 30–60 min in the light, and a third one that maintains a high level after 1 h of transfer to darkness (Gill et al., 2002). The FTR–TRX system responds rapidly to light after transfer from darkness, while expression of trxC and trxQ genes is less pronounced after light transfer, since expression of both genes is relatively unaffected in the dark (Figure 2). It is worth noting that the FTR genes together with trxA and trxB genes decrease their expression (15–10% of the original level) upon shifting to darkness for 1 h (Figure 2B). Hence, FTR–TRX genes behave like PSI and phycobilisome genes with respect to light–dark transitions (Gill et al., 2002). The fact that the half-lives of the different transcripts are unaffected by light or darkness (Supplemental Figure 1) clearly indicates that transcription rate is affected by the changes from light to darkness.
The effect of the PET inhibitors DCMU and DBMIB, on the transcript levels of FTR–TRX genes, indicated again a decrease in the expression of these genes, except for trxC and trxQ (Figure 3). A similar study was carried out in Chlamydomonas reinhardtii showing that mRNA levels of the chloroplastic Trx m, but not of the cytosolic Trx h decreased in DCMU or DBMIB-treated cultures (Lemaire et al., 2002). We have previously shown that several Synechocystis genes, such as glnA and glnB (Garcia-Dominguez and Florencio, 1997; Reyes and Florencio, 1995), and the trxA gene (Navarro et al., 2000) are regulated by the photosynthetic electron flow or by glucose metabolism. A genome-wide transcript analysis showed that the phycobilisome subunits, the photosystems I and II, and the ATP synthase genes are affected negatively by both inhibitors and support the idea that all these genes are under the control of the redox state of electron transport components other than the plastoquinone (PQ) pool (Hihara et al., 2003). However, many other genes respond differently to DCMU or DBMIB, indicating that there are genes that probably respond to the PQ redox state. Interestingly, a putative NADP-thioredoxin reductase (NTR) gene (slr0600) is expressed at higher levels in the presence of both inhibitors (Hihara et al., 2003), whereas the ftr genes decrease (Figure 3), suggesting that the putative NTR may be the reducing system for TrxC and TrxQ under these conditions. Furthermore, trxA expression was strongly affected by DBMIB, compared to the other thioredoxin genes (Figure 3), and, again, trxC and trxQ gene expression was independent of the presence of these inhibitors whereas ftr genes decrease under these conditions (Figure 3). The fact that glucose clearly attenuated the effect of DCMU and, to a lesser extent, DBMIB suggests that the redox-sensor controlling transcription of the FTR–TRX genes (Figure 4) could be situated after the PQ pool. Despite the fact that our study demonstrated that the expression of the FTR–TRX genes is regulated via photosynthetic conditions, the amount of the corresponding proteins might be unaffected, at least at moderate times, as it has been reported that, in Chlamydomonas cultures treated with heavy metals, the Trx m RNA levels increased, whereas the amounts of Trx m protein remained unaffected (Lemaire et al., 1999).
Functional Role of TrxB and TrxQ
One of the areas of great interest in thioredoxin studies is to establish the role of each thioredoxin in the different photosynthetic organisms and a classical approach is to obtain mutants of specific thioredoxin genes and analyze the corresponding phenotype. The existence in Synechocystis of one copy of each of four different thioredoxin types makes this organism an ideal model to study this issue compared with higher plants, where certain redundancy exists, such as for h- or m-type thioredoxins. Moreover, insertional Synechocystis mutants are relatively easy to obtain, whereas construction of mutants of the green alga Chlamydomonas is less straightforward (Buchanan and Balmer, 2005; Florencio et al., 2006; Lemaire et al., 2007; Lemaire and Miginiac-Maslow, 2004; Meyer et al., 2005). Three cyanobacterial thioredoxins (TrxA, m-type; TrxB, x-type, and TrxQ, y-type) have homologs in the chloroplast, suggesting that some roles could be conserved (Florencio et al., 2006; Hisabori et al., 2007). Here, we have focused on mutation and segregation of the genes encoding the TrxB and TrxQ thioredoxins, since it was previously reported that a null mutant of trxA cannot be obtained in Synechocystis unless another trxA gene is introduced in the cell (Navarro and Florencio, 1996). Thus, STXB, STXQ, and the double mutant STXQB strains generated were viable under normal growth conditions (Figure 6), suggesting a non-essential role of these proteins in Synechocystis. However, STXB is affected in the adaptation to high light, indicating that TrxB may play a role in this adaptation, probably by sensing an excess of reducing conditions that triggers a response to this stress. The finding that this strain was also sensitive to DTT (Figure 6C) supports the view of a role for this thioredoxin under reducing conditions. Currently, there are no data about Trx x mutants in other photosynthetic organisms in order to compare this phenotype. On the other hand, STXB strain is resistant to diamide (Figure 6C). The hyper-tolerance to this compound of the TrxB mutant was previously described for strains lacking TRX2 gene in Saccharomyces cerevisiae, which may arise as the result of activation of some component of the oxidative stress in this mutant (Muller, 1996). The STXQ mutant exhibits a weak phenotype in regard to conditions that provoke oxidative stress, such as hydrogen peroxide treatment, which has been shown to up-regulate trxQ expression in genome-wide transcript analysis in Synechocystis (Kanesaki et al., 2007; Li et al., 2004).
Biochemical studies have shown that higher plant Trx x and Trx y are very efficient as electron donors to 2-Cys peroxiredoxin and peroxiredoxin Q, respectively (Collin et al., 2003; Rouhier et al., 2004). In Synechocystis, TrxB was also reported to reduce type II peroxiredoxin, but at low efficiency (Hosoya-Matsuda et al., 2005). Taken together, these data strongly suggest a role for both thioredoxins in the oxidative stress response, although a more detailed and specific study would be required.
TrxBC34S from SBC34S Cells Is a Non-Functional Version of TrxB Thioredoxin
Growth of SBC34S mutant cells is similar to the one of STXB strain under both photoautotrophic conditions and reducing or oxidative stress (Figure 6C). This result is somehow expected, since TrxBC34S is a non-functional TrxB thioredoxin, namely it is able to form complexes with its targets but cannot reduce these targets properly because of the absence of the second cysteine in the active site. Therefore, both cysteines (CXXC) are absolutely necessary for thioredoxin function.
Advantages of the In-Vivo Approach Developed to Identify Target Proteome of TrxB
In this study, we have developed a strategy to identify targets of TrxB in vivo. This is one of the few in-vivo analyses developed up to now to identify putative targets of thioredoxins, together with the two-hybrid study to identify thioredoxin targets in Arabidopsis (Vignols et al., 2005), since the majority of them have been in-vitro approaches (Balmer et al., 2003; Hosoya-Matsuda et al., 2005; Lemaire et al., 2004; Lindahl and Florencio, 2003; Mata-Cabana et al., 2007; Perez-Perez et al., 2006; Yamazaki et al., 2004).
This approach was carried out in a mutant strain lacking functional TrxB (STXB), in order to avoid competition between the wild-type version of the protein and TrxBC34S. Furthermore, the STXB strain still contains other thioredoxins, such as TrxA, TrxC, and TrxQ, and the analysis is thus developed in a physiological context. However, the fact that other thioredoxins are active makes it more difficult to obtain putative targets because, probably, the disulphide bond formed between TrxBC34S and its target is broken by other thioredoxins or reductant before complex purification. This handicap will be very difficult to solve in photosynthetic organisms where many isoforms and types of thioredoxins and glutaredoxins are present in the same cellular compartment. The situation is easier in non-photosynthetic organisms where thioredoxin and glutaredoxin number is limited (Lemaire et al., 2007).
Another advantage of our approach is that we have introduced a Synechocystis thioredoxin into a Synechocystis strain, namely it is not a heterologous system, as previously reported for plant thioredoxins introduced in yeast (Issakidis-Bourguet et al., 2001; Mouaheb et al., 1998). In our case, the redox cascade can be maintained without alterations.
The putative targets identified in Table 1 revealed a number of proteins previously described in the in-vitro studies in Synechocystis (Lindahl and Florencio, 2003; Perez-Perez et al., 2006) as well as new proteins. One reason for the loss of specificity among different thioredoxins might be that the mutation of one cysteine renders the Trx mutant more reactive towards various targets rather than others. Among new targets, it is worth noting glutamine synthetase, which was found in proteomic studies in higher plants and Chlamydomonas (Balmer et al., 2003; Lemaire et al., 2004; Marchand et al., 2004; Motohashi et al., 2001; Yamazaki et al., 2004) but not in Synechocystis (Lindahl and Florencio, 2003; Perez-Perez et al., 2006). However, no redox regulation has been reported for this enzyme in Synechocystis (Garcia-Dominguez et al., 1999). Fructose, 1-6,bisphosphatase (FBPase), another classical target of thioredoxins in photosynthetic organisms, was also identified in our in-vivo study, despite the fact that no redox regulation has been reported about this enzyme in cyanobacteria (Tamoi et al., 1998). Moreover, FBPase was absent from our previous in-vitro studies in Synechocystis, indicating that our system could find targets that in-vitro approaches could not retain.
Another interesting result was the identification of several ribosome proteins from the small and the large subunits (Table 1), which might be related to an association of TrxB with the control of translation in Synechocystis according to growth conditions.
In this regard, it has been shown in yeast that thioredoxins are required for protection against stress induced by DTT (Trotter and Grant, 2002). More recently, in similar studies, strains deleted for Gpx3 (a glutathione peroxidase), Tsa1 (a 2-Cys peroxiredoxin), and Skn7 (a transcription factor involved in oxidative stress response) were found to be sensitive to DTT (Rand and Grant, 2006). Thioredoxins act upstream of Tsa1 in the thioredoxin-dependent DTT resistance pathway. A mutant lacking the active site cysteine residues was unable to rescue the DTT sensitivity of a tsa1-deficient strain, indicating that peroxiredoxin enzyme activity is required under these conditions. Tsa1 plays a role as an antioxidant protein in the detoxification of hydrogen peroxides, but, more recently, it has been shown to act as a molecular chaperone that promotes resistance to heat shock (Jang et al., 2004). Tsa1 may therefore protect against reductive stress via its peroxidase or chaperone activities. TrxB from Synechocystis, as shown for x-type thioredoxin from photosynthetic eukaryotes (Collin et al., 2003), may act as an electron donor for 2-Cys Prx, and both proteins may play a role in the reductive stress response in the cyanobacterium Synechocystis.
In summary, our results show that the FTR–TRX system in Synechocystis is regulated by the photosynthetic conditions via the photosynthetic electron flow, except two of thioredoxin genes (trxC and trxQ) that apparently escape this control. This indicates the existence of a link between photosynthesis and the redox state exerted via thioredoxin in the control of the many metabolic processes in cyanobacteria. The proteomic in-vivo analysis of the TrxB targets identifies new protein targets, especially those ribosomal proteins that may link thioredoxin with a coordinated control of the translation processes in cyanobacteria.
Further efforts would be required in the near future to establish the relationship between changes in the redox state of cyanobacteria and the specific response of the thioredoxin system.
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Cyanobacterial Strains and Growth Conditions
Synechocystis sp. PCC 6803 cells were grown photoautotrophically at 30°C in BG11C medium (Rippka et al., 1979) supplemented with 1 g l–1 NaHCO3 and bubbled with a continuous stream of 1% (v/v) CO2 in air under continuous illumination (50–70 µE m–2 s–1). Mutant strains were grown under the same conditions but supplemented with the appropriate antibiotic: spectinomycin at a final concentration of 5 µg ml–1 (Synechocystis STXB), kanamycin at a final concentration of 50 µg ml–1 (Synechocystis STXQ) or with both antibiotics (STXQB, SBC34S).
In order to analyze the effects of high light intensity, Synechocystis sp. PCC 6803 was grown under 500 µE m–2 s–1 illumination. Oxidative stress conditions were set up by addition of 1 mM hydrogen peroxide to cultures in the exponential-growth phase. For cells grown on plates, BG11C liquid medium was supplemented with 1% (w/v) agar. Different diamide concentrations (0–500 µM) were added to BG11C medium to trigger oxidative stress. A reducing stress was induced by adding dithiotreitol (0–20 mM).
To analyze trx and ftr gene expression under different photosynthetic conditions, the photosynthetic inhibitors DCMU (10 µM) or DBMIB (10 µM) were added to the medium. In mixotrophic cultures, BG11C medium was supplemented with 10 mM glucose. Heterotrophic growth was achieved by covering cultures with sheets of aluminium.
Sequences of Genes and Proteins
Gene and protein sequences were obtained from the Cyanobase (http://bacteria.kazusa.or.jp/cyanobase/). We named the genes: trxA (slr0623), trxB (slr1139), trxC (sll1057), and trxQ (slr0233) for thioredoxin genes, and ftrC (sll0554) and ftrV (ssr0330) for catalytic and variable ferredoxin–thioredoxin reductase genes, respectively.
Target Disruption of the ORFs trxB and trxQ
A 663-bp DNA fragment containing trxQ was amplified by PCR from Synechocystis sp. PCC 6803 genomic DNA using the oligonucleotides TrxQFor (5'-ACTGGATTAATATCTTTGCG-3') and TrxQRev (5'-GAACCGAAAGCTTTTTCTGG-3'). The amplified fragment was cloned into pBlueScript II SK (+)(Stratagene). The CK1 cassette (Kmr) was excised from pRL161 (Elhai and Wolk, 1988)) by digestion with HincII and cloned into the RsaI site of trxQ. The resultant plasmids were designated pTrxQKm+ and pTrxQKm– and included CK1 cassette in positive or negative orientation, respectively. Synechocystis cells were transformed with these plasmids to generate the STXQ mutant strains.
In a similar way, a 958-bp DNA fragment containing trxB was amplified by PCR using primers TrxBFor (5'-GCTTTTATGTGCTCACGGGC-3') and TrxBRev (5'-CAACCGCAAGAGAATTTCCA-3'). The amplified fragment was cloned into pBlueScript II SK (+). The streptomycin/spectinomycin cassette (Spr/Spr) was obtained from pHP45
(Prentki and Krisch, 1984) by digestion with BamHI and was inserted into the BamHI site of trxB. The resultant plasmids were named pTrxBSp+ and pTrxBSp– and included Spr in positive or negative orientation, respectively. Both plasmids were used to transform WT and STXQ mutant Synechocystis cells to generate STXB and STXQB mutant strains, respectively.
Transformed cells were selected on agar-solidified BG11C medium supplemented with 5 µg ml–1 spectinomycin (Synechocystis STXB), 50 µg ml–1 kanamycin (Synechocystis STXQ) or with both antibiotics (STXQBSTXBQ). Complete segregation of mutants was confirmed by Southern blot analysis using trxB or trxQ probes.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from 35-ml samples of Synechocystis cultures at mid-exponential phase (3–5 µg ml–1 chlorophyll). Cyanobacterial cells were grown in the presence of compounds that cause alterations in the photosynthetic electron flux such as DCMU, DBMIB, or glucose. RNA extraction was performed by vortexing cells in the presence of phenol/chloroform and acid-washed baked glass beads (0.25–0.30 mm diameter, Sigma Aldrich) as previously described (Garcia-Dominguez and Florencio, 1997). For Northern blotting, 15 µg of total RNA was loaded per lane and electrophoresed on denaturing formaldehyde-containing 1.5% agarose gels. Transfer to nylon membranes (Hybond N-plus, GE Healthcare), prehybridization, hybridization, and washes were performed as recommended by the manufacturer. Probes for the genes were obtained by PCR. As a control, the filters were reprobed with a 580-bp HindIII and BamHI probe from plasmid pAV1100 containing the constitutively expressed RNase P RNA gene (rnpB) from Synechocystis (Vioque, 1992). Relative transcripts levels were quantified with a scanning densitometer (Cyclone Phosphor System, Packard Instrument) or an Instantimager Electronic Autoradiograph (Packard Instrument).
Determination of mRNA Half-Lives
To determine the effect of light and darkness on trx and ftr transcripts stability, the RNA synthesis was blocked by addition of rifampicin (400 µg ml–1) to the mid-exponential phase cultures and total cellular RNA was isolated as indicated above. The decay of transcript was followed by dot-blot hybridization. After quantitation of mRNA accumulation, the half-life of the transcript was estimated from a plot of relative transcript level versus time.
Anti-TrxB Antibody Production and Western Blot Analysis
Anti-TrxB antisera were obtained according to standard immunization protocols by injecting purified TrxB protein in rabbit. For Western blot analysis, proteins were fractionated in 12 or 15% SDS–PAGE according to the method of Laemmli (1970) and immunoblotted with anti-TrxB (1:2.000) or anti-HIS (1:1.000) (Sigma Aldrich) antibodies. The ECL Plus immunoblotting system (GE Healthcare) was used to detect TrxB with anti-rabbit or His-tagged TrxB with anti-mouse secondary antibodies (Sigma Aldrich).
Generation of the Synechocystis SBC34S Strain
In order to obtain the in-vivo target proteome of the Synechocystis thioredoxin TrxB, we generated a His-tagged cysteine-to-serine mutant strain (SBC34S) where the cysteine 34 of TrxB was replaced by a serine residue. This strain does not express the wild-type version of the trxB gene. To this end, we introduced in Synechocystis STXB cells the mutant version of trxB (trxBC34S) under the control of the Trc promoter. The gene was introduced into the nrsD gene interrupted with a CK1 cassette. The resultant plasmid was called pBC34S and was used to transform Synechocystis STXB cells. The correct integration and complete segregation of the mutant strain were tested by Southern blotting.
Purification of the Putative TrxBC34S-Target Complexes
Cytosolic proteins were obtained from Synechocystis SBC34S cultures growing photoautotrophically in the exponential-growth phase. Before harvesting the cells, cultures were transferred to darkness for 2 h to stop the photosynthetic electron flux. Thereafter, cells were broken by sonication in 50 mM Tris-HCl (pH 8.0) buffer and the soluble fraction was separated by centrifugation, first at 20 000 rpm for 15 min and then the supernatant was further centrifuged at 45 000 rpm for 45 min. Then, soluble proteins were incubated with a His-bind Ni-affinity resin (Novagen, CN Biosciences Nottingham, UK) for 1–2 h at 4°C under gentle agitation. Matrix was washed first with 50 vol. of buffer A (50 mM Tris-HCl (pH 8), 300 mM NaCl) and then with the same volume of buffer B (50 mM Tris-HCl (pH 8), 300 mM NaCl, 50 mM imidazole). Finally, intact thioredoxin-target mixed disulphide complexes were eluted with buffer C (50 mM Tris-HCl (pH 8), 300 mM NaCl, 1 M imidazole). All steps of this procedure were carried out at 4°C.
Separation of Target Proteins
1-D and 2-D non-reducing/reducing SDS–PAGE for optimal overall resolution of thioredoxin and its target proteins was performed as described elsewhere (Lindahl and Florencio, 2003; Perez-Perez et al., 2006). Twelve percent acrylamide gels for the first dimension and 10 or 12% acrylamide gels for the second dimension were performed. Instead of normal conditions, 0.2% SDS-containing gels and 7 M urea-containing solubilization buffer were used in order to increase protein solubility. Following separation, gels were silver-stained by using ‘Silver staining kit’ (GE Healthcare), or subjected to analysis by immunoblotting with anti-TrxB or anti-HIS antisera.
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SUPPLEMENTARY DATA |
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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 supported by grants from the Ministry of Science and Education (BFU2004–00050 and BFU2007–60300/BMC) to F.J.F., and Junta de Andalucia (BIO284 and CVI-099) to F.J.F.
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Acknowledgements |
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We thank Anna M. Lindahl and José L. Crespo for a critical reading of the manuscript. M. Esther Peréz-Peréz was recipient of a fellowship (FPI) from the Ministry of Science and Education. No conflict of interest declared.
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