JXB Advance Access originally published online on November 10, 2005
Journal of Experimental Botany 2005 56(422):3193-3206; doi:10.1093/jxb/eri316
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RESEARCH PAPER |
Bioinformatic analysis of the genomes of the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 for the presence of peroxiredoxins and their transcript regulation under stress
1Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany
2Molecular Cell Physiology, Faculty of Biology, University of Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany
* To whom correspondence should be addressed. Fax: +49 521 106 6039. E-mail: karl-josef.dietz{at}uni-bielefeld.de
Received 12 May 2005; Accepted 16 September 2005
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Abstract |
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The genomes of the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 encode five and six open reading frames (ORFs), respectively, with similarity to peroxide-detoxifying peroxiredoxins (Prx). In addition to one highly conserved gene each for 2-Cys Prx and 1-Cys Prx, the Synechocystis sp. PCC 6803 genome contains one TypeII Prx and two PrxQ-like ORFs, while Synechococcus elongatus PCC 7942 has four PrxQ-like ORFs. The transcript regulation of all these bioinformatically identified genes was analysed under selected stress conditions, i.e. light limitation and light stress, hydrogen peroxide, methylviologen, salinity, as well as nitrogen- and iron-deficiency. The results on specific time- and stress-dependent regulation of transcript amounts suggest conserved as well as variable functions of these putative Prx-s in antioxidant defence. The results are discussed in the context of evolution and physiological function, particularly in relation to photosynthesis.
Key words: Oxidative stress, peroxiredoxins, photosynthesis, Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionNote added in proofReferences |
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Cyanobacteria, being the first photosynthetic organisms with an oxygenic type of photosynthesis, utilizing water as an electron donor and producing dioxygen as a by-product, represent the link between heterotrophically growing bacteria and photosynthetic eukaryotes (Whitton and Potts, 2000
). In the same way as other oxygenic photosynthetic organisms, cyanobacteria face the problem of capturing light energy efficiently, while minimizing oxidative damage caused by a surplus of absorbed energy, especially under conditions when the anabolism is slowed down, as, for example, due to nutrient limitation (Huner et al., 1996; Schwarz and Forchhammer, 2005). Therefore, it can be assumed that cyanobacteria were among the first organisms to evolve effective mechanisms for protection from oxidative stress (see reviews by Barber and Andersson, 1992; Aro et al., 1993; Anderson et al., 1995; Mittler, 2002).
In cyanobacteria, the ROS-scavenging systems include enzymes such as superoxide dismutases, catalases, peroxidases, and peroxiredoxins (see reviews by Regelsberger et al., 2002; Dietz et al., 2005). The unicellular mesophilic cyanobacterium Synechocystis sp. PCC 6803 possesses one superoxide dismutase encoded by the gene slr1516. This cyanobacterium also contains one catalase (sll1987) which belongs to the catalase-peroxidase type (Tichy and Vermaas, 1999; Mutsuda et al., 1996). However, in contrast to plants, Synechocystis sp. PCC 6803 does not seem to contain a higher plant-type ascorbate peroxidase, which is present in chloroplasts as thylakoid-bound and stromal isoforms, and is considered to be the most crucial peroxidase in chloroplasts, detoxifying, in combination with superoxide dismutase, the superoxide anion formed at the acceptor side of photosystem I (Foyer et al., 1994; Asada, 1999). In Synechocystis sp. PCC 6803, in contrast to the situation in eukaryotes, the Mehler reaction of photosystem I does not seem to produce ROS. The O2 is photoreduced by (an) A-type flavoprotein(s) directly to water by electron transfer from photosystem I (Helmann et al., 2003). Thus, the mechanism of scavenging the reactive oxygen species formed under stress at the acceptor side of photosystem I is different in plants compared with cyanobacteria (at least in the cyanobacteria investigated so far). Obviously, it must have been an advantage to replace these efficient A-type flavoproteins by another detoxification system after the oxygen concentration had increased in the atmosphere and after compartmentation of the photosynthetic and respiratory apparatus into chloroplasts and mitochondria had taken place.
Synechocystis sp. PCC 6803 only contains one superoxide dismutase and one catalase-peroxidase, but possesses five genes encoding peroxiredoxins (Prx-s) (Kobayashi et al., 2004; Dietz et al., 2005; Hosoya-Matsuda et al., 2005). Prx-s are a ubiquitous family of antioxidant enzymes which have been identified in eubacteria, archaea, yeast, algae, higher plants, and animals (Dietz, 2003; Wood et al., 2003b). All Prx-s share the same basic catalytic mechanism, in which an activated cysteine (the peroxidative cysteine) is oxidized to a sulphenic acid by the peroxide substrate. They have a rather low activity, but possess a broad substrate specificity. Possible substrates are hydrogen peroxides, alkyl hydroperoxides, and peroxynitrites. Reductive regeneration of the oxidized catalytic thiol depends on glutathione, thioredoxin, glutaredoxin, cyclophilin, and tryparedoxin. Prx activity can be regulated in vivo by cysteine oxidation, aggregation state, phosphorylation, or limited proteolysis. These regulatory mechanisms have, so far, mainly been investigated in eukaryotes. Moreover, evidence has recently been presented that some Prx-s have a function as regulators of redox-mediated signal transduction at least in some eukaryotes (Hofmann et al., 2002; Dietz, 2003; Wood et al., 2003a; Veal et al., 2004) besides having a function in scavenging peroxides. Therefore, Prx-s are important components of the cellular antioxidant defence system as well as in redox homeostasis.
In plants, Prx proteins are categorized into four subclasses based on subunit composition, number and location of the conserved cysteine residues as well as the sequence environment of the catalytic centre. They also show differences with respect to the reductant which is predominantly used.
- (i) 2-Cys Prx-s contain two conserved cysteines and are homodimeric enzymes where the two subunits interact in the catalytic cycle and are linked via a disulphide bond in the oxidized form. Moreover, the enzyme can undergo redox-sensitive oligomerization. Eukaryotic 2-Cys Prx-s not only act as antioxidants, but are also shown to regulate H2O2-mediated signal transduction. 2-Cys Prx-s exclusively localize to the chloroplast (Baier and Dietz, 1997; Motohashi et al., 2001).
- (ii) 1-Cys Prx-s contain a single conserved catalytic cysteine and are preferentially expressed in plants in the embryo and aleurone (Stacy et al., 1996
). The catalytic cycle is not fully understood.
- (iii) Prx-s typeII (atypical 2-Cys Prx-s) can use thioredoxin and glutaredoxin as the reductant and can exist in multiple isoforms localized in plants in many subcellular compartments, including one in plastids (PrxIIE; Horling et al., 2002
).
- (iv) Prx-sQ (atypical 2-Cys Prx-s) are homologues of the E. coli bacterioferritin co-migrating protein and function as monomers (Kong et al., 2000
). In plants, PrxQ is imported by chloroplasts.
- (ii) 1-Cys Prx-s contain a single conserved catalytic cysteine and are preferentially expressed in plants in the embryo and aleurone (Stacy et al., 1996
) as well as Synechocystis sp. PCC 6803 (Tichy and Vermaas, 1999). Most likely the cell is not able to provide enough reducing equivalents to degrade large amounts of hydrogen peroxide rapidly via Prx-s, or their catalytic efficiency with less than 200 mol substrate mol–1 enzyme min–1 is too low. Based on the evaluation of the genomes of three marine cyanobacteria having a rather small genome (see CyanoBase: http://www.kazusa.or.jp/cyano), it has also been suggested that the cyanobacteria Synechococcus strain WH8102 as well as the two Prochlorococcus marinus strains MI9313 and MED4 do not possess a gene(s) with homology to any catalase, but possess several sequences with homology to Prx-s (Perelman et al., 2003) and CyanoBase: http://www.kazusa.or.jp/cyano). These two findings might suggest that the Prx-s were probably the early enzymes to scavenge hydrogen peroxide, and the catalase-type enzymes gained a new function in the cells when the O2 concentration in the atmosphere increased and, as a consequence, the ROS formation in the cell under stress also went up. However, the reason for the existence of multiple genes for Prx-s in Synechocystis sp. PCC 6803 and other cyanobacteria with no significant compartmentation is still unknown.
Since the majority of cyanobacteria (possibly all of them), whose genomes have been sequenced, have more than one Prx, a two-step approach was undertaken in this work to improve the knowledge on Prx-s. First, in a bioinformatic approach, the Prx-s present in Synechocystis sp. strain PCC 6803, which was the first cyanobacterium whose genome was sequenced (Kaneko et al., 1996), and in Synechococcus elongatus PCC 7942, whose genome has recently been sequenced (DOE Joint Genome Institute: http://genome.ornl.gov/microbiol/syn_PCC7942), were identified and compared with those of Arabidopsis thaliana (The Arabidopsis Genome Initiative 2000: http://www.arabidopsis.org/). Second, the expression pattern of the Prx-s in both cyanobacteria was investigated by transcript analysis under selected growth conditions.
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Materials and methods |
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Cyanobacterial strains and growth conditions
Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 were obtained from the Pasteur Culture Collection of Cyanobacterial Strains, Paris, France. The cells were grown in gas wash-bottles of 250 ml in a stream of 2% (v/v) CO2-enriched air and in BG11 medium according to Rippka et al. (1979)
with slight modifications as given in Stephan et al. (2000). The bottles were placed in a water bath of 30 °C. Inoculation was done with cells to result in an OD750nm=0.35. In general, the cells were grown for 72 h. If modified, conditions are listed in the legends of the figures. For most experiments, the cells were illuminated from the top with four Philips lamps (120 W, PAR 38 EC cool beam) at a distance of 35 cm from the water bath surface resulting in a light intensity of 200 µE m–2 s–1 measured as total quantum flux density between 400 and 740 nm on the water surface with a Quantaspectrometer QSM-2500 (Techtum Instrument, Umea, Sweden). In the experiments, in which the effects of different light intensities were compared, the above light intensity of 200 µE m–2 s–1 (referred to as medium light intensity) was used and, in addition, a light intensity referred to as high or low light intensity. The high light intensity was obtained by using three Philips lamps which were placed at the side of the water bath at a distance of 10 cm from the culture bottles resulting in a light intensity of 800 µE m–2 s–1. To avoid overheating the water bath, the air in front of the bath was circulated by ventilation. Low light intensity corresponding to 20 µE m–2 s–1 was obtained by using two neon tubes (Sylvania Luxline ES 18 W Warmton, Osram, Munich, Germany) placed at the side of the water bath at a distance of 45 cm from the culture bottles.
For growing the cells in nutrient-limiting BG11 media, the cells were harvested by centrifugation (15 min at 3500 r.p.m.), washed once with distilled water, and resuspended in the corresponding media: iron limitation (30 µM iron-III-citrate completely omitted) and nitrate limitation (17.65 mM NaNO3 completely omitted, the medium contained in addition 20 mM 4-(2-hydroxyethyl) piperazine-1-propanesulphonic acid (EPPS)-NaOH, pH 7.5). Salt stress was achieved by adding 0.6 M NaCl to the BG11 medium.
Growth was determined by measuring the absorbance of Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 cultures at 750 nm.
Treatment of the cyanobacteria with hydrogen peroxide or methylviologen
Synechocystis PCC 6803 or Synechococcus elongatus PCC 7942 cells were cultivated for 48 h with an illumination of 200 µE m–2 s–1. To such a culture 5 mM hydrogen peroxide or 50 µM methylviologen was added and growth was continued under the same conditions. Cells were harvested at the times given in the corresponding figures.
Isolation of total RNA from Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942 and northern blot analysis
Cyanobacterial cells were cultivated for four cycles, with another dilution before the experiments for harvesting the cell samples for RNA isolation were started. The cyanobacterial cultures were collected at different times during regular growth in BG11 medium or media as indicated. After centrifugation of 40 ml cell culture in 50 ml tubes with crushed ice at 3500 r.p.m. for 10 min (Heraeus Sepatech Megafuge 1.0) the cell sediments were immediately frozen in liquid nitrogen. Until further analysis the samples were stored at –75 °C or –20 °C.
Total RNA was isolated using a hot acidic phenol extraction procedure (Reddy et al., 1990) and the RNeasy Kit (Qiagen, Hilden, Germany). The aqueous phase resulting from the phenol extraction was applied to spin-through columns of the kit for further purification. For hybridization experiments, 5 or 10 µg RNA were denatured at 60 °C in a formaldehyde/formamide-containing buffer and separated in a formaldehyde-containing 1.3% agarose gel (Michel et al., 1999). For rnpB, isiA, slr1198, and sll1621 of Synechocystis sp. PCC 6803, as well as for rnpB, gene 915, 782 and 662 of Synechococcus elongatus PCC 7942, 5 µg of total RNA were loaded. For all other genes (sll0755, slr0242, and sll0221 of Synechocystis sp. PCC 6803 as well as for the genes 310, 439, and 1668 of Synechococcus elongatus PCC 7942), 10 µg of total RNA were loaded. After capillary-transfer to Hybond N+ membranes (Amersham Pharmacia Biotech, Freiburg, Germany) RNA was UV-crosslinked to the membrane and samples were probed with different PCR-derived digoxygenin-11-dUTP labelled (DIG-dUTP) gene-specific probes. PCR with specific primers (Table 1) was carried out using the Taq polymerase kit (Qiagen) and substitution of 1/40 to 1/10 for Synechocystis sp. PCC 6803 and 1/20 for Synechococcus elongatus PCC 7942 of the regular dTTP concentration with DIG-dUTP (Roche Molecular Biochemicals, Mannheim, Germany). The rnpB probe was used in all experiments to ensure equal loading of total RNA. Blots were processed according to the manufacturer's recommendation. Northern blots with hydrogen peroxide-treated samples were performed three times, while all other northern blots were performed twice. All the figures compare representative northern blots selected from independent experiments.
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Database search and sequence evaluation
Utilizing the derived protein sequences of the ten genes encoding peroxiredoxins of Arabidopsis thaliana (The Arabidopsis Genome Initiative 2000), the genome of Synechocystis sp. strain PCC 6803 (CyanoBase, Kaneko et al., 1996
) was searched for proteins representing Prx-s. All promising candidate proteins of putative Prx-s were verified by alignment with the protein sequence of Prx-s from several organisms (but mainly Arabidopsis thaliana) in ClustalW. Subsequently, the draft genome sequence of Synechococcus elongatus PCC 7942 (DOE Joint Genome Institute: http://genome.jgi-psf.org/microbiol/) was searched with the sequences of the ten Prx-s of Arabidopsis thaliana and the five Prx-s sequences from Synechocystis sp. PCC 6803. Again classification as Prx-s was done by comparing the derived protein sequences in ClustalW. Protein sizes as number of amino acids, molecular masses, and pI values were calculated utilizing the ProtParamTools of the ExPASy-Proteomics Server under www.expasy.org/tools/protparam.html. Values for identity/similarity were obtained by using the program BlastP and the Matrix BLOSUM62 under www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi. |
Results |
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Bioinformatic analyses of the genome sequences of Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 for the presence of peroxiredoxin encoding genes
In the model plant Arabidopsis thaliana ten genes encoding Prx-s, which can be assigned to the four Prx classes, have been identified (Dietz et al., 2002
). These are one 1-Cys Prx, two 2-Cys Prx-s, six TypeII Prx-s, and one PrxQ. The molecular masses of these Prx proteins vary from about 17 kDa of the cytosolic TypeII Prx-s to about 29 kDa for the chloroplastic 2-Cys Prx A and B (including signal sequence). Only the TypeII Prx A is different in size and structure from all other Arabidopsis thaliana Prx-s, because its derived protein sequence consists of two parts and it has a predicted size of about 63 kDa. Most likely, this gene is a pseudo Prx gene. The theoretical pIs of the Arabidopsis thaliana Prx-s are in the acidic region for most of them, except for the PrxQ which, even after removal of the signal sequence, has a pI in the basic region (Table 2).
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Analysing the genome sequence of Synechocystis sp. PCC 6803 (Kaneko et al., 1996
) revealed the presence of five Prx-s (Kobayashi et al., 2004; Hosoya-Matsuda et al., 2005). For evaluation, proteins were selected which have similarity to peroxiredoxins, but proteins which lack the catalytic cysteine or have a typical glutaredoxin domain (as for example Sll1159) were excluded. The five Prx-s in Synechocystis sp. PCC 6803 are one 1-Cys Prx, one 2-Cys Prx, one TypeII Prx, and two PrxQ. The molecular masses of the derived proteins range from 17.6 (Slr0242) to 23.6 kDa (Slr1198), and the pI is acidic for all of them. The highest similarity of 82% exists between the 2-Cys Prx (Sll0755) of Synechocystis sp. PCC 6803 and the 2-Cys Prx A and B of Arabidopsis thaliana (Fig. 1). The similarity of the other four Synechocystis sp. PCC 6803 Prx-s to the corresponding Arabidopsis thaliana proteins are between 51–66% (Table 2).
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Analysing the genome of Synechococcus elongatus PCC 7942, whose sequence just recently became available, led to the identification of six putative Prx-s genes. These are one 1-Cys Prx, one 2-Cys Prx, and four PrxQ. The molecular masses range from 15.7 kDa (Gene662) to 21.8 kDa (Gene782), and again the pI is acidic for all of them. As in Synechocystis sp. PCC 6803, the highest similarity of 84% and 83% exists between the 2-Cys Prx (Gene782) and the 2-Cys PrxA and PrxB of Arabidopsis thaliana, respectively (Fig. 1). The similarity of the five other Prx-s to the corresponding Arabidopsis thaliana proteins lies between 54–71% (Table 3).
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Comparing Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, the results reveal that both strains possess one gene encoding a 1-Cys Prx (Slr1198 and Gene915, respectively). The similarity between these two genes of 90% (identity 83%) is very high. The two strains also contain one 2-Cys Prx each (Sll0755 and Gene782, respectively), and again the similarity between the two derived proteins is very high (88% similarity and 73% identity). However, in contrast to Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942 contains no TypeII Prx. Another difference is that Synechococcus elongatus PCC 7942 contains four PrxQ-s, while Synechocystis sp. PCC 6803 only contains two PrxQ-s (Table 4). Three of the PrxQ-s in Synechococcus elongatus PCC 7942 (genes 310, 439, and 662) possess the second cysteine in the conserved amino acid sequence region, and both cysteines are spaced by only a few amino acids which is typical for the eukaryotic Q-type Prx-s. By contrast, the gene 1668 of Synechococcus elongatus PCC 7942 and the two Prx-s of Synechocystis sp. PCC 6803 (Sll0221 and Slr0242) which possess similarity to Q-type Prx-s, lack the second cysteine in the conserved amino acid region and thus represent atypical PrxQ-type enzymes (Fig. 2). When comparing the Prx-s family of Arabidopsis thaliana with the two cyanobacteria investigated here, it can be stated that all three organisms possess one 1-Cys Prx. The two cyanobacteria contain one 2-Cys Prx whereas Arabidopsis thaliana has two 2-Cys Prx-s. Arabidopsis thaliana has multiple TypeII Prx-s, while Synechocystis sp. PCC 6803 only encodes one TypeII Prx and Synechococcus elongatus PCC 7942 no TypeII Prx. By contrast, the two cyanobacteria contain multipe type-Q Prx-s (two and four for Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, respectively), while Arabidopsis thaliana only has one PrxQ that functions in the context of photosynthesis (Horling et al., 2003
; Lamkemeyer et al., 2003).
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Comparative analyses of the mRNA transcript pools of peroxiredoxins in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 under selected stress conditions
The transcript level of the five Prx-s in Synechocystis sp. PCC 6803 and the six Prx-s in Synechococcus elongatus PCC 7942 were investigated under the standard growth condition and under growth conditions which induce oxidative stress. The latter include hydrogen peroxide and methylviologen treatment as well as growth under high light intensity, iron limitation, and NaCl stress. In addition to the mRNA level for the various Prx-s, in some experiments the expression levels of the isiA and the isiA/B transcripts, which have previously been shown to be up-regulated under iron limitation and oxidative stress (Michel and Pistorius, 2004
) as well as under NaCl stress (Vinnemeier et al., 1998), were also investigated. The gene isiA encodes a chlorophyll a binding protein which forms a new antenna around photosystem I (Bibby et al., 2001; Boekema et al., 2001), and the gene isiB encodes the FMN-containing flavodoxin which can, in part, replace the iron–sulphur-containing ferredoxin (Straus, 1994). The transcript levels of the five Prx-s of Synechocystis sp. PCC 6803 and the six Prx-s of Synechococcus elongatus PCC 7942 were investigated by northern blot analysis after cultivation with light of 20, 200, or 800 µE m–2 s–1 intensity and referred to as low, medium, and high light intensity. The growth curves are presented in Fig. 3. In Synechocystis sp. PCC 6803, when grown under low light intensity, practically no mRNA of any of the Prx-s was detectable, except a very low level of the mRNA for 2-cys prx. By contrast, under medium light intensity, the transcripts encoding 1-cys prx, 2-cys prx, typeII prx, and prxQ-B1 were higher than under low light. The mRNA level was especially high in the initial growth phase after dilution of the cells into fresh medium (growth for 6 up to 24 h) and was lower in the later growth phase (growth for 24 up to 48 h). The transcript for prxQ-B2 was not or hardly detectable. The mRNA levels of these Prx-s under high light were comparable to those under medium light (Fig. 4).
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In a converse manner, in Synechococcus elongatus PCC 7942, substantial expression of most Prx-s transcripts was already observed under low light (Fig. 5). The mRNA pools for 1-cys prx, 2-cys prx, and prxQ-A1 were up-regulated and the mRNA for 2-cys prx and 1-cys prx were positively responsive to dilution at low light (Fig. 5B). Under medium light (Fig. 6) all Prx-s transcripts were present at an elevated level, but the highest up-regulation was seen for 2-cys prx and prxQ-A1. Thus, one major difference between the two species was that in Synechococcus elongatus PCC 7942 the transcripts for some Prx-s were already up-regulated under low light, while in Synechocystis sp. PCC 6803 up-regulation of most Prx transcripts required a substantially higher light intensity.
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Since it is well documented that excess light causes oxidative stress (Niyogi, 1999
), the expression levels of the Prx transcripts in both cyanobacterial strains were investigated under oxidative stress imposed by treatment with hydrogen peroxide or with methylviologen. The mRNAs of isiA and isiAB that are known to be up-regulated in response to oxidative stress (Yousef et al., 2003) were taken as a control. Their stimulated transcript levels confirmed the establishment of oxidative stress (Figs 7, 8). Under hydrogen peroxide stress, typeII prx showed the highest up-regulation among all Prx-s in Synechocystis sp. PCC 6803 (Fig. 7). The up-regulation was even higher when cells were treated with methylviologen (Fig. 8). This is in good agreement with published results (Kobayashi et al., 2004; Hosoya-Matsuda et al., 2005, and references therein). By contrast with the up-regulation of the 2-cys prx transcript under hydrogen peroxide, the 1-Cys Prx transcript level was lower under hydrogen peroxide and methylviologen stress than under regular growth condition. In Synechococcus elongatus PCC 7942 the transcript level of 2-cys prx was strongly up-regulated under hydrogen peroxide as well as under methylviologen stress (Figs 7, 8, right-hand side). The transcript level for this Prx was much higher than for any of the other Prx-s, confirming previously published results by Perelman et al. (2003).
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In response to treatment with 0.6 M NaCl, growth of Synechocystis sp. PCC 6803 was greatly reduced while growth of Synechococcus elongatus PCC 7942 almost completely ceased (Fig. 9). The results of Fig. 10 show that the isiA and isiA/B transcripts increased slightly within 24 h and strongly within 48 h in Synechocystis sp. PCC 6803, similar to previous reports (Vinnemeier et al., 1998
). In Synechocystis sp. PCC 6803 four Prx-s were up-regulated with distinct time dependencies, i.e. either passing through a maximum after 24 h (2-cys prx, prxQ-B1), being similarly expressed after 24 and 48 h (typeII prx) or further increasing from 24 to 48 h (1-cys prx). In Synechococcus elongatus PCC 7942 prxQ-A2, 1-cys prx, and 2-cys prx were highly up-regulated under NaCl stress, while prxQ-A3 and prxQ-B were also up-regulated, but less than 1-cys prx and 2-cys prx. At equal NaCl concentration, salt stress was more severe in Synechococcus elongatus PCC 7942 than in Synechocystis sp. PCC 6803 (see growth curves of Fig. 9). This is in agreement with the higher level of 1-cys prx and 2-cys prx transcript expression (Fig. 10). The results indicate that hydrogen peroxide, methylviologen, and salt stress mainly affect expression of 1-cys prx, 2-cys prx, and typeII prx. However, a difference was observed with regard to which of the Prx-s had the highest up-regulation. In Synechocystis sp. PCC 6803 under hydrogen peroxide and methylviologen stress, the mRNA of typeII prx was highly induced; more so under methylviologen stress than under hydrogen peroxide stress. Salt stress caused only a minor expression of this Prx. In Synechococcus elongatus PCC 7942 under hydrogen peroxide and methylviologen stress, the mRNA of 2-cys prx was mainly up-regulated, while under salt stress the mRNA of 1-cys prx and 2-cys prx were strongly increased.
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A rather complex interrelationship exists between iron deficiency and oxidative stress (Michel and Pistorius, 2004
). Therefore, the influcence of iron limitation on prx expression was also investigated (Fig. 11). Unexpectedly, only a minor up-regulation of Prx transcripts was observed even after prolonged iron deficiency. In Synechocystis sp. PCC 6803 2-cys prx and prxQ-B1 transcripts showed a slight increase. However, the mRNA of typeII prx, which was up-regulated under oxidative stress caused by hydrogen peroxide or methylviologen exposure, was non-responsive. In Synechococcus elongatus PCC 7942 the prxQ-A1 transcript was mainly increased. It seems that the levels of 1-cys prx and 2-cys prx transcripts being present in iron-sufficient cells were high enough to protect the cells under iron limitation and that the up-regulation of one of the Q-type Prx-s was sufficient for further protection.
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In addition, nitrate limitation was chosen to investigate whether a lack of a major nutrient would lead to a general or a specific up-regulation of prx transcripts. For Synechocystis sp. PCC 6803 as well as Synechococcus elongatus PCC 7942 nitrate limitation caused an increased expression of 2-cys prx, while the expression of 1-cys prx decreased under nitrate limitation (not shown). An increase of expression on protein level for the 2-cys prx has already been described for Synechococcus elongatus PCC 7942 by Schwarz and Forchhammer (2005)
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionNote added in proofReferences |
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This publication provides the first complete comparative account of prx genes in two cyanobacterial species, Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, and presents comprehensive data on prx expression in response to a set of environmental cues. Since cyanobacteria define the oldest evolutionary group of photoautotrophic organisms with an oxygenic-type of photosynthesis, the enzymatic equipment including Prx-s may provide clues to the evolution of photosynthesis and antioxidant defence. Both cyanobacterial genomes encode one 1-Cys Prx with 63% and 68% similarity at the amino acid level to the corresponding 1-Cys Prx in Arabidopsis thaliana. In higher plants, the 1-Cys Prx has a dual cellular location in the cytosol and nucleus, and is preferentially found in seed tissue (Stacy et al., 1996
), but also in vegetative tissue from the desiccation-tolerant species Xerophyta viscosa (Mowla et al., 2002). Similarly to the suggested function of 1-Cys Prx in plants, it may be hypothesized that the cyanobacterial 1-Cys Prx is involved in protecting nucleic acids from oxidative damage, particularly under stress. With the exception of hydrogen peroxide- and methylviologen-induced oxidative stress, 1-cys prx mRNA increased in response to all kinds of metabolic imbalances, here, for instance, imposed by changing irradiance, salinity, and iron deficiency. Heterologously expressed Slr1198 only had a low peroxidase activity. However, disruption of the gene significantly reduced the growth rate of the Synechocystis sp. PCC 6803 cells (Hosoya-Matsuda et al., 2005). The barley 1-Cys Prx PER1 was the first plant Prx for which a protective property to DNA breakage was demonstrated in a mixed function oxidation assay containing Fe2+, O2, and DTT (Stacy et al., 1996).
The cyanobacterial 2-Cys Prx-s are highly conserved with 82% to 84% similarity to the corresponding 2-Cys PrxA and PrxB of Arabidopsis thaliana. Its higher plant counterpart has a defined function in photosynthesis. Depending on its redox related oligomeric state, the 2-Cys Prx cycles between a thylakoid-bound and stromal state (Konig et al., 2002). Thus, in addition to a role in peroxide detoxification and as alternative mechanism to ascorbate peroxidase-mediated detoxification of Mehler reaction-derived hydrogen peroxide (Dietz et al., 2002), a redox signalling function of 2-Cys Prx has been proposed in the context of photosynthesis (Dietz, 2003; Dietz et al., 2005). In cyanobacteria, 2-cys prx mRNA levels were highly responsive to any type of metabolic disturbance, i.e. light, methylviologen, hydrogen peroxide, NaCl, iron, and nitrogen starvation. The results contrast the rather constitutively high level of expression in photosynthesizing plant cells with only a little up-regulation upon oxidative stress, but a strong down-regulation in response to decreased electron pressure at photosystem I (Baier et al., 2004) and feeding of reducing agents (Baier and Dietz, 1996; Horling et al., 2001). In prokaryotes, 2-Cys Prx are suggested to detoxify peroxide substrates generated at low rates in regular undisturbed metabolism (Wood et al. 2003a, b). Sequence comparisons of 2-Cys Prx from Synechocystis sp. PCC 6803, the red alga Porphyra, where the 2-Cys Prx is still encoded in the chloroplast genome, and the higher plant 2-Cys Prx has led to the hypothesis that the 2-Cys Prx originates from the former cyanobacterial endosymbiont (Baier and Dietz, 1997). Later during evolution, the 2-Cys Prx was transferred from the plastome to the nuclear genome under N-terminal fusion of a suitable transit peptide (Baier and Dietz, 1997). In agreement with that hypothesis, the transit peptide is encoded by a separate exon (Baier and Dietz, 1997). From the data presented here, it is suggested that the 2-Cys Prx had already acquired a function in protecting oxygenic photosynthesis in cyanobacteria, and evolved additional features in redox signalling in plants. From the expressional data it may be concluded that the protective role is essential whenever the conditions for photosynthesis are sub-optimal. The essential role of Synechocystis sp. PCC 6803 2-Cys Prx (Sll0755) in photosynthetic adaptation has been established in a deletion mutant that showed increased stress sensitivity and a disturbed peroxide detoxification metabolism (Klughammer et al., 1998; Yamamoto et al., 1999). Moreover, from gene disruption analysis in Synechococcus elongatus PCC 7942 it was concluded that the Prx, which has high similarity to Sll0755 of Synechocystis sp. PCC 6803 (88% similarity of gene 782 to Sll0755), is also essential for growth during excessive radiation and that under such conditions the mutant strain could not compete with the wild type (Perelman et al., 2003).
The Synechocystis sp. PCC 6803 genome codes for a TypeII Prx that had highest identity with the plastidic PrxII E of Arabidopsis thaliana. PrxII E has been described as a house-keeping Prx of the plastids that is not related to photosynthesis, but rather involved in general plastid metabolism (Horling et al., 2003; Perelman et al., 2003; Dietz et al., 2005). Synechocystis sp. PCC 6803 typeII prx–mRNA was strongly up-regulated upon treatment with methylviologen, hydrogen peroxide and, to a lesser extent, in response to light, salt, iron, and nitrogen deprivation. In the latter case, the increase was transient. DNA microarray analysis of Synechocystis sp. PCC 6803 has already revealed the strong up-regulation of the transcript of the gene sll1621 encoding the TypeII Prx in response to hydrogen peroxide (Li et al., 2004) and also upon challenging the cells with methylviologen (Kobayashi et al., 2004). Moreover, it was shown that a Fur-type transcription factor (Slr1738) plays a regulatory role in the induction of sll1621 in response to oxidative stress (Kobayashi et al., 2004; Li et al., 2004) and that the expression of sll1621 seems to be redox regulated (Hihara et al., 2003). However, it should also be mentioned that, on protein level, no remarkable changes for Slr1198 and Sll1621 were detected under hydrogen peroxide (Hosoya-Matsuda et al., 2005). Genetic disruption of sll1621 indicated that the gene product is essential for aerobic phototrophic growth, especially in high light. Hosoya-Matsuda et al. (2005) have investigated the two Prx-s Sll1621 (TypeII Prx) and Slr1198 (1-Cys Prx) after isolating the proteins by thioredoxin affinity chromatography. Sll1621 has a high glutathione-dependent peroxidase activity. Again, disruption of gene sll1621 had a dramatic effect on the viability of the Synechocystis sp. PCC 6803 cells even under weak light conditions. Interestingly, despite the essential feature of the TypeII Prx in Synechocystis sp. PCC 6803, a homologous gene was absent from the Synechococcus elongatus PCC 7942 genome. It is tempting to speculate that one or several of the four Q-type Prx-s substitute(s) for the TypeII Prx which was lost during evolution in Synechococcus elongatus PCC 7942. From the expressional pattern, PrxQ-B or partly PrxQ-A1 resembled most TypeII Prx in its response to hydrogen peroxide, methylviologen and salinity.
PrxQ is present as a single gene in Arabidopsis thaliana (Horling et al., 2002, 2003). Surprisingly, PrxQ-like proteins constitute the largest Prx sub-families both in Synechocystis sp. PCC 6803 with two isogenes and Synechococcus elongatus PCC 7942 with four isogenes. In Synechocystis sp. PCC 6803, PrxQ-B2 was hardly expressed under any condition tested. In Synechococcus elongatus PCC 7942, the four prxQ genes showed a time dependency and stress-specific pattern of expression. This is most obvious in the hydrogen peroxide experiment (Fig. 7). Kinetics of induction and the time point of maximal expression differed for each of the four PrxQ isoforms. The similarity of the cyanobacterial PrxQ-isoforms varied between 53% and 76% and was not much different between the cyanobacterial and higher plant PrxQ. PrxQ of higher plants functions in the context of photosynthesis. It has been localized to the chloroplast (Lamkemeyer et al., 2003; Rouhier et al., 2004) and is up-regulated upon oxidative stress, particularly after adding hydrogen peroxide to leaf slices, but also after administration of diamide to oxidize the glutathione pool and of t-butylhydroperoxide as well as after transfer to high light (Horling et al., 2003). In a pathogen–plant interaction, prxQ transcripts rapidly and strongly accumulated in leaves (Rouhier et al., 2004). In the same study, from six tested thioredoxins (from heterologous systems) the cytosolic Trx h3 most efficiently donated electrons to oxidized PrxQ. Glutaredoxin was ineffective. In another study using homologous partners, from all the tested chloroplastic thioredoxins, Trx y1 and y2 were the most efficient electron donors for plastidial PrxQ (Collin et al., 2004). PrxQ also associates with the thylakoid membrane from Arabidopsis thaliana (P Lamkemeyer, H Li, K-J Dietz, unpublished results). All these data support the conclusion that the plant PrxQ functions in the context of antioxidant defence and in the redox homeostasis of photosynthesis.
In eukaryotes, due to an often encountered post-transcriptional regulation, transcript levels do not immediately and necessarily convert into corresponding protein amounts. A tighter relationship between transcript and protein amounts is expected in prokaryotes. Based on that simplified assumption, 2-Cys Prx appears to be the predominant Prx in Synechococcus elongatus PCC 7942, but also Q-type Prx, particularly PrxQ-A1, may be abundant antioxidants. The identification of 2-Cys Prx in a global proteomics approach by Fadi Aldehni et al. (2003) and Schwarz and Forchhammer (2005) confirms that this particular Prx can accumulate to significant amounts in nitrogen-starved Synechococcus elongatus PCC 7942. In chloroplasts, 2-Cys Prx is present in the range of 0.6% of soluble protein (Konig et al., 2003). By contrast, the TypeII Prx appears predominant in Synechocystis sp. PCC 6803. This allows another conclusion that relates to the contribution of peroxiredoxins to antioxidant defence in photosynthesis on an evolutionary scale. Apparently, peroxiredoxins have acquired specific and indispensable functions; the function of 2-Cys Prx and 1-Cys Prx may be conserved, whereas other Prx may replace each other as suggested above for TypeII Prx in Synechocystis sp. PCC 6803 and PrxQ-B and possibly PrxQ-A1 in Synechococcus elongatus PCC 7942. Complementation of knock-out lines may provide clues to this end.
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The draft version of the genome of Synechococcus elongatus PCC 7942 of 21 June 2005 presented an altered gene nomenclature.
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The work originates from a joint collaboration of TP3 and TP7 within the special research focus of the DFG FOR 387 (Redox regulation in photosynthesis). Support within Di 346/6 is also acknowledged.
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Abbreviation: Prx, peroxiredoxin.
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