Identification of the proteomic changes in Synechocystis sp. PCC 6803 following prolonged UV-B irradiation

Abstract

The diversified physiological responses in cyanobacteria under ultraviolet-B (UV-B) radiation have been broadly researched. The changes in the metabolic control mechanisms hidden behind these physiological traits still need to be further investigated. This research attempts to identify some of the internal mechanisms of several stressful phenotypes such as a decreased growth rate, an impaired photosystem, and the degradation of photosynthetic pigments. Different expression levels of proteins in the cytoplasm of Synechocystis sp. PCC 6803 under short-term and long-term UV-B stress were investigated by using a comparative proteomic approach. One hundred and twelve differentially expressed protein spots were identified by mass spectrometry to match 75 diverse protein species. They mainly focus on amino acid biosynthesis, photosynthesis and respiration, energy metabolism, protein biosynthesis, cell defence, and other functional groups. By focusing on these areas, the study reveals the correlation between UV-B stress-responsive proteins and the physiological changes listed above. The research, showing that short-term response-proteins are quite different from long-term response-proteins, helps to identify the change in homeostatic mechanisms in Synechocystis sp. PCC 6803. Related putative functions of these proteins and the physiological responses of cyanobacteria under UV-B stress, a UV-B responsive protein network in Synechocystis sp. PCC 6803 under long-term stress was successfully produced. Such a protein network helps to increase our understanding of the comprehensive functional network cyanobacteria use to adapt to UV-B stress. In addition, 30 novel proteins not previously found related to UV-B stress were identified. This opens up new areas for exploration to identify the response to UV-B stress in cyanobacteria.

2-D gel electrophoresis, proteome, Synechocystis, UV-B stress

Introduction

The enhanced levels of solar UV-B radiation reaching the surface of the Earth has become an important issue over the past two decades. The first reports of damage to the stratospheric ozone layer raised many new questions. (Crutzen, 1992; Smith et al., 1992). Enhanced UV-B radiation adversely affects terrestrial photosynthetic organisms and is also known to penetrate aquatic environments fairly deeply (Smith et al., 1992; Bernal et al., 2005).

Cyanobacteria, the widespread and abundant oxygenic photosynthetic prokaryotes, make a large contribution to phytoplankton primary productivity over large oceanic regions (Richardson and Jackson, 2007). Like most other phototrophic organisms, cyanobacteria must withstand the detrimental effects of solar UV-B due to their dependence on light for survival.

It should also be taken into consideration that, especially in early Precambrian (1.5 Ga ago), higher UV irradiation occurred and that the future UV regime will increase according to the reasons previously mentioned. To some extent, early organisms must have been adapted to high UV irradiation. Therefore, an understanding of UV adaptation may be gained by investigating the oldest oxygenic organisms, such as cyanobacteria (Castenholz and Garcia-Pichel, 2000).

Among cyanobacteria, extensive research shows that UV-B has a number of negative effects on cell physiology, damaging nucleic acids, proteins, and lipids (Unsal-Kacmaz et al., 2002). UV-B photons are also known to cause important damage to the photosynthetic apparatus (Teramura, 1980; He and Hader, 2002).

Until now the molecular mechanisms of diversified physiological response in cyanobacteria under UV-B stress have not been fully identified. Somewhat surprisingly, only a few studies have been conducted on this important topic (Balogi et al., 2008; Kos et al., 2008). Past research mainly focused on the effects of UV-B on the photosynthetic apparatus and PSII/D1(Vass et al., 2005).

The best characterized photoautotrophic micro-organism related to UV stress response is the cyanobacterium Synechocystis sp. PCC 6803. Studies using this cyanobacterium are greatly enhanced by the complete sequencing of its genome (Kaneko et al., 1996). Global gene expression profiling experiments have shown that genes involved in a variety of cellular processes were affected by just 2 h of UV-B stress (Huang et al., 2002). Although large-scale transcriptome analysis has documented the (transcriptional) dynamics of a large number of antioxidative genes, the mRNA abundance is not always consistent with the protein level (Gygi et al., 1999). Moreover, previous research has indicated that the analysis of differential gene expression is actually an indirect approach to understanding the molecular mechanisms involved in the stress-response (Fulda et al., 2006; Kurian et al., 2006). Proteomics can be used to bring the virtual life of genes to the real life of proteins. Physiological proteomics will generate a new and broad understanding of cellular physiology because the majority of proteins synthesized in the cell can be visualized (Hecker and Volker, 2004). Clearly, it is essential to utilize proteomic and even metabolic strategies to gain a system-level understanding of cyanobacterium responses to UV-B stress (Lei et al., 2005).

In the present work, the first functional proteomic investigation of cytoplasmic proteins that are responsive to UV-B in Synechocystis sp. PCC 6803 was initiated. Using two-dimensional electrophoresis (2-DE) in combination with MS/MS analysis, proteins were characterized whose expression is altered upon exposure to short-term and long-term UV-B stress. Such a UV-B stress challenges results in a dramatic proteomic response in at least 112 protein spots. Identification of these proteins, their abundant changes, and the impact of UV-B stress on cyanobacterium photosynthesis all reveal a close link between the changes in the abundance of specific proteins. They also highlight the overall defence response to short-term and long-term UV-B stress, and give a global view of the ubiquitous cellular changes under UV-B stress. This helps to establish the first possibly interconnected protein network induced by UV-B in Synechocystis sp. PCC 6803. The results presented in this paper provide the framework for further functional studies of each member of this network in the adaptive mechanisms of UV resistance of cyanobacteria. It is hoped that this research may also be used to assist in the further understanding of higher plants.

Materials and methods

Organism and stress treatment

A glucose-tolerant strain of wild-type Synechocystis sp. PCC 6803 was grown in 4.0 l borosilicate glass at 30 °C under constant illumination from incandescent lamps (L 40 W/25 S, Osram, approximately 170 μmol photons m⁻² s⁻¹) in BG-11 medium (Rippka et al., 1979), The experiment started when the culture was in the exponential growth stage, at almost 10⁶ cells ml⁻¹. Each culture was equally divided into 24 parts. Twelve of them were treated with UV-B. The selected periods of UV radiation are 8 h, 72 h, 84 h, and 96 h. Eight hours represents the beginning of the exponential growth phase; 72–96 h are from the end of the exponential growth phase to the beginning of the stationary growth phase. Each treatment group included three replications. The other 12 were grouped in threes without UV-B treatment and used as controls. All of the cultures were placed in 250 ml UV- transparent quartz Erlenmeyer flasks (the transmittance of UV-B is larger than 95%) and closed with sterile cotton stoppers which allowed gas exchange. Visible light was obtained from incandescent lamps as the radiation source for growth. Additional UV-illumination at 1 W m⁻² intensity was provided by a Philips TL 40 W/12 lamp with maximal emission at 310 nm. The bulbs were covered using cellulose acetate (CA) filters to exclude wavelengths lower than 280 nm. As a control, cyanobacteria were exposed for the same period of time under the same lamps covered with polyester (PE) (no UV-B treatment). The UV-B irradiance was measured by a UV radiometer which was new and calibrated at the factory; the radiometer had specific UV sensors sensitive at 310 nm (Beijing Electro-Optical Equipment Factory, China). The depth of samples was just 1 cm, so the attenuation was very small. The irradiance of transmitted light was almost equal to the incident intensity. All samples were gently agitated by bubbles produced by aeration flow during irradiation to ensure uniform distribution. Aeration flow was kept at 1:1 vvm by bubbling air at atmospheric pressure. Each treatment was replicated three times. All of the samples (treated and controls) were grown in the same thermo-constant chamber conditions. The only difference being the exposure to UV-B irradiation.

Growth rates, pigments, and chlorophyll fluorescence measurements

Samples were taken from each radiation treatment daily to determine cell concentration. Cell concentrations were obtained by microscope-counting. Immediately after sampling, cells were fixed with 0.2% glutaraldehyde (Sigma-Aldrich, St Louis, MO) for 30 min at 4 °C. Fresh medium was added to reach the desired cell concentration for microscope-counting.

Chlorophyll a (Chl a) was determined from the absorbance of the methanol extracts at 666 nm (Mackinney, 1941).

For the determination of carotenoids, samples were harvested by centrifugation, and the pellet was saponified by suspension in 30% (v/v) methanol containing 5% (w/v) KOH. The remaining pellet was neutralized by the addition of 70% (v/v) acetic acid, and carotenoids were then extracted by the addition of pure dimethylsulphoxide and maintained at 70 °C for 5 min. The absorbance of the supernatant was measured at 490 nm and the concentration of carotenoids was calculated using the specific absorption coefficient ϵ%=2200 (Davies, 1976).

The optimal quantum yield (F_v/F_m) and other fluorescence parameters were determined using a portable pulse amplitude modulated fluorometer (PAM – Water-ED, Walz, Germany). The efficiency of excitation capture by open PSII centres (F_v^′/F_m^′) was calculated as (F_m^′– F_o^′)/F_m^′, which is also referred to as the efficiency with which excitation energy is transferred to open PSII centres. The photochemical quenching coefficient qP was defined as (F_m^′–F_s)/(F_m^′– F_o^′). PSII quantum efficiency was (F_v^′/F_m^′)qP=( F_m^′–F_s)/F_m^′(Genty et al., 1989).

Protein extraction

For each control and treatment, at least three protein samples were prepared, representing at least three biological replicates of the experiment. Cytoplasmic protein samples were prepared by the method described by Kurian et al. (2006). Cells were harvested from control as well as treated samples. Whole cell lysates were prepared by resuspending harvested cells in 10 mM HEPES-NaOH (pH 7.2) supplemented with 1 mM PMSF as protease inhibitor. Cells were broken by vortexing with glass beads (0.17–0.18 mm) at 4 °C. Glass beads and unbroken cells were removed by centrifugation (4000 g) for 10 min. The cytoplasmic proteins were in the supernatant. Salts and insoluble impurities were removed using a 2-D Clean-Up Kit (GE Healthcare). Protein concentrations were estimated using the Bio-Rad protein assay (Bio-Rad).

2-DE, gel staining, and image analysis

Prior to IEF, the samples were diluted with the rehydration buffer (RB: 6 M urea, 2 M thiourea, 2% CHAPS, and 40 mM DTT) so as to load 1.1 mg protein per 24 cm immobilized polyacrylamide gel (IPG) strip (4–7). The strips were subjected to active rehydration at 30 V h⁻¹ for 18 h on an Ettan IPGphor system (GE Healthcare) with the following IEF program: 200 V for 40 min, 500 V for 40 min, 1000 V for 1 h, 4000 V for 2 h, and 8000 V for ∼8.5 h, until 70 000 VhT was achieved. For the second dimension, the strips were equilibrated in two steps and electrophoresed by using an Ettan Dalt Six multiple-gel electrophoresis unit (GE Healthcare). Three gel replicates were run for each triply-extracted sample. A total of six gels (three treated and three control samples) were run simultaneously to ensure maximum reproducibility of the results. Proteins spots were visualized by the Blue Silver (modified CBB staining) method (Candiano et al., 2004).

The gels were scanned with UMAX PowerLook 2100XL scanner (Willich, Germany) using LabScan 5.0 software and the image analysis was done by Image Master 2D Platinum software 5.0 (GE Healthcare). Three separated gels for each sample were treated as a replicate group. Matching spots were rechecked manually. Spot volume was taken as a percentage relative to the total volume of all the spots in the gel. The relative volume of each spot was assumed to represent its expression level. A criterion of P <0.05 was used to define the significant difference when analysing the parallel spots between groups with one-way ANOVA and the Student–Newman–Keuls test using the SAS software package version 8.2 (SAS Institute).

In-gel tryptic digest

The protein spots were excised from the gel, subjected to in-gel digestion and MALDI samples of the extracted peptides were prepared as described by Kurian et al. (2006).

MALDI-TOF/TOF MS and MS/MS analysis and database search

The peptides were diluted with 0.8 μl matrix solution [α-cyano-4-hydroxy-cinnamic acid (CHCA, Sigma, St Louis, MO, USA) in 0.1% trifluoroacetic acid (TFA), 50% acetonitrile (ACN)] before being spotted on to the target plate. Samples were allowed to air-dry before being analysed by a 4700 MALDI-TOF/TOF Proteomics Analyser (Applied Biosystems, Foster City, CA, USA). The UV laser was operated at a 200 Hz repetition rate with a wavelength of 355 nm. The accelerated voltage was operated at 20 kV. Myoglobin digested by trypsin was used to calibrate the mass instrument with the internal calibration mode. All acquired spectra of samples were processed using 4700 ExploreTM software (Applied Biosystems) in a default mode. Parent mass peaks with range 700–3200 Da and minimum S/N 20 were picked out for tandem TOF/TOF analysis. Combined MS and MS/MS spectra were submitted to MASCOT (V2.1, Matrix Science, London, UK) by GPS Explorer software (V3.6, Applied Biosystems) and searched with the following parameters: NCBInr database (release date: 2006.03.18), taxonomy of bacteria, trypsin digest with one missing cleavage, none fixed modifications, MS tolerance of 0.2 Da, MS/MS tolerance of 0.6 Da, and possible oxidation of methionine. Known contaminant ions (keratin) were excluded. A total of 4 736 044 sequences and 1 634 373 987 residues in the database were actually searched. MASCOT protein scores (based on combined MS and MS/MS spectra) of greater than 72 were considered statistically significant (P ≤0.05). The individual MS/MS spectra with statistically significant (confidence interval >95%) best ion scores (based on MS/MS spectra) were accepted. To eliminate the redundancy of proteins that appeared in the database under different names and accession numbers, the single-protein member belonging to the species Gallus or with the highest protein score (top rank) was singled out from the multi-protein family.

Results

Physiological responses in Synechocystis sp. PCC 6803 under UV-B stress

The growth profile of Synechocystis in response to UV-B stress is shown in Fig. 1A. It was evident that, under UV-B stress, the growth rate of treatments of Synechocystis sp. PCC 6803 was roughly 60% less than the rate of the controls. After 4 d of UV-B treatment, the amount of chlorophyll a of Synechocystis sp. PCC 6803 was reduced by 60.4% compared to controls (Fig. 1B). The carotenoid content of Synechocystis did not decrease as much as chlorophyll a. The declining ratio compared to controls was 25% (Fig. 1C). After 4 d of UV-B treatment at 1 W m⁻², there was a progressive reduction of PSII quantum efficiency in the treatments of Synechocystis compared to the control cultures. The maximal decreasing ratio that appeared on the third day was almost 70%. On the fourth day, the PSII quantum efficiency increased 10% compared to the third day (Fig. 1D).

Fig. 1.

Effects of UV-B stress on the growth (A), chlorophyll a content (B), carotenoid content (C), and the optimal quantum yield (F_v/F_m) (D) in Synechocystis sp. PCC 6803. The changes in 4 d under 1W m⁻² UV-B radiation were measured. Data are expressed as mean values ±SD (n=3). (Open square), control; (closed square), treatment.

Synechocystis sp. PCC 6803 proteome in response to UV-B stress

Using the extraction protocol in combination with CBB staining, the average number of detectable spots in this study reached approximately 1000 on each 2-DE gel. This suggests that full advantage could be taken of the proteomic strategy to obtain more abundant information about the effects of UV-B stress on cyanobacteria. Figure 2 shows six reproducible gel maPSIn accordance with three controls and three corresponding treatments (8, 72, 96 h). From a spot-to-spot comparison and statistical analysis, the differentially expressed protein spots whose expression level was more than 1.5 times higher or lower in treated gels compared to the corresponding controls (P ≤0.05), were subjected to protein identification. Measurement was done by mass spectrometry analysis as described in the Materials and methods. Interpretable MS/MS spectra were obtained for 112 of the 128 spots that were differentially expressed.

Fig. 2.

CBB G-250 stained 2-DE gel images (pH range 4–7) comparing soluble proteins from the cytoplasmic fraction of Synechocystis grown under control and and the corresponding UV-B treatment.

Among the 112 identified proteins, 66 were up-regulated and 46 were down-regulated in response to UV-B stress (see Supplementary Tables S1 and Supplementary Data at JXB online). Most of these spots had greater than a 1.5-fold change in abundance under at least one of the UV-B treatments, and 64 of these spots exhibited more than a 2-fold change (see Supplementary Tables S3 and Supplementary Data at JXB online). The stress response of a bacterial cell following an environmental signal can be divided into a short-term response and a long-term response (Panoff et al., 1997). An example of a short-term response used 28 proteins, of which 12 were up-regulated and 16 were down-regulated, and occurred within the first 8 h (Fig. 2A). The second part consisted of a long-term response of 84 proteins, of which 54 were up-regulated and 30 were down-regulated (Fig. 2B). This long-term stress required from 3–4 d, the stress-time lasted until the first part of the stationary growth phase (Fig. 1A).

Our results showed that 14 unipros appeared as 54 identities (see Supplementary Tables S1 and Supplementary Data at JXB online).This can occur because more than one member of a gene family is regulated by UV-B radiation. It is also possible because there is post-translational modification of a single gene product upon UV-B exposure. Interestingly, ten unipros representing 26 identities (isoforms) showed that each set of isoforms had the same up- or down-regulated change patterns in abundance in response to UV-B stress. At the same time, the other four unipros [glutamate–ammonia ligase, phycocyanin associated linker protein, phosphate transport ATP-binding protein, and methionine sulphoxide reductase A (Msr-A)] appeared as 28 isoforms that exhibited opposite expression patterns within each set of isoforms (Fig. 2; see Supplementary Tables S1 and Supplementary Data at JXB online). Likewise, many similar phenomena were observed in previously reported proteomic studies (Casati et al., 2005; Fulda et al., 2006). Based on changes in the isoelectric state of protein spots on 2-DE gels with a phosphatase treatment, Casati et al. found that the up-regulated or down-regulated expression of pyruvate, phosphate dikinase by UV-B radiation were regulated by phosphorylation and dephosphorylation, and a different Thr residue appears to regulate the activity of this enzyme by UV-B radiation (Casati et al., 2005). In this research, it was found that, when exposed to UV-B, Msr-A which PI changed to nearly 4.4 in 2D-gels was increased in its expression level and the PI which matched its theoretical PI (5.77) showed a decrease in the expression level (see Supplementary Tables S1 and Supplementary Data at JXB online). Post-translational modifications of Msr-A might be a possible cause. This finding provided reasonable speculation that proteomic responses of the latter four unipros were probably involved in post-translational regulation, such as phosphorylation and de-phosphorylation. The post-translational modification of these unipros in response to UV-B stress might result in the opposite expression patterns of different isoforms with a phosphorylation or de-phosphorylation state. These results suggest that isoforms of certain unipros may play the same or different roles in modulating defence responses to UV-B stress in Synechocystis sp. PCC 6803. Post-translational modification of proteins is one likely strategy involved in cyanobacterial responses to UV-B stress.

Identification and functional classification of the differentially expressed proteins

Figure 3A summarizes the key findings in cyanobacterial processes responding to UV-B treatments based on the identification of representative proteins. They were sorted according to the functional categories defined by CyanoBase (http://www.kazusa.or.jp/cyanobase/Synechocystis/ index.html). All of the identities were classified into 17 major categories (Fig. 3A). An impressive 74% of these identified proteins were implicated in seven functional groups, including amino acid biosynthesis, photosynthesis and respiration, energy metabolism, translation, cellular processes, hypothetical, biosynthesis of cofactors and prosthetic groups. One aim of this work was to identify proteins quantitatively affected by short- and long-term UV-B radiation. The difference of the short-term response-proteins and the long-term response-proteins is shown in Fig. 3B and C. One interesting phenomenon is that the number of changed short-term response-proteins is significantly larger than the long-term response-proteins, while the number of changed short-term response-proteins is smaller. The changed short-term response-proteins belong to cell envelope, translation, transcription, DNA replication etc whereas long-term response-proteins are involved in regulatory functions, photosynthesis and respiration, biosynthesis of cofactors and prosthetic groups.

Fig. 3.

Functional classification and distribution of identified proteins. (A) Functional classification of all proteins. (B) Functional classification of proteins of 8 h UV-B stress. (C) Functional classification of proteins of 3–4 d UV-B stress.

Differentially expressed novel UV-B responsive proteins including the hypothetical and unknown group

Thirty novel proteins, which had never been reported in current research related to UV-B stress, were discovered in this work (Table 1). Identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in the acclimation of cyanobacteria under UV-B stress. Some of them belong to the hypothetical and unknown group (Table 1). 2-D PAGE coupled to MS/MS will help to corroborate or disprove the annotation of the genome sequence by providing evidence for the presence of proteins previously classified as hypothetical. Despite similarities to proteins from other organisms, the cellular function of hypothetical proteins in relation to UV-B stress is not clear. Some proteins seem to be unique for Synechocystis (spot 028, spot 070, spot 112), since none of the other fully sequenced cyanobacteria or other organisms has a gene homologous to them. Generally, the abundance of these two grouPSIs rather low compared with that of those proteins whose function is already known (Panoff et al., 1997). The identification of a large number of hypothetical proteins is particularly interesting because a preliminary prediction of their function may be feasible. Their classification as members of the UV-B stress stimulus means they are most likely to be involved in the adaptation to UV-B stress in cyanobacteria (Hecker and Volker, 2004). Monitoring the true expression of hypothetical or conserved hypothetical proteins is the first step toward their function and cellular localization. Their regulation and possible roles under this stress will be the focus of future work.

Table 1.

List of the differentially expressed novel proteins identified by MALDI-TOF/TOF

Proteome of long-term UV-stress cells

For better understanding of the mechanisms of how cyanobacteria survive and grow under long-term UV-B stress, the differentially expressed proteins were carefully checked under long-term UV-B stress. Among the 84 long-term response-proteins identified in Synechocystis sp. PCC 6803, down-regulated proteins were preferentially associated with three functions; amino acid biosynthesis, photosynthesis, and protein biosynthesis (see Supplementary Table S2 at JXB online; Fig. 4). In the functional group of amino acid biosynthesis, five of the seven identified unipros were decreased in their expression levels. These proteins included 3-phosphoshikimate 1-carboxyvinyltransferase (spot 030), cysteine synthase (spot 031), tryptophan synthase subunit beta (spot 032), glutamate–ammonia ligase (spot 034) and sulphate adenylyltransferase (spot 041). 3-phosphoshikimate 1-carboxyvinyltransferase and sulphate adenylyltransferase had never been reported to be related to UV-B stress. Since exposure to UV-B stress led to an impaired photosynthetic function of Synechocystis sp. PCC 6803, a clear down-regulation of spots for 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (spot 072), 1-deoxy-D-xylulose-5-phosphate synthase (spot 073), phosphoribulokinase (spot 074), phycocyanin b subunit (spot 080), and phycocyanin associated linker protein (spot 082) were reproducibly found (71% in identified unipros of photosynthesis). Spot 072 and spot 073 are essential enzymes in the mevalonate-independent pathway of isoprenoid biosynthesis (Souret et al., 2002; Kemp et al., 2003; Bernal et al., 2005). Carotenoids and the phytyl side chain of chlorophylls are isoprenoids derived from this pathway. Spot 074 is a thiol-regulated enzyme of the Calvin cycle. Spot 080 and spot 082 are light-harvesting proteins. The mRNA amount of the two corresponding genes was also reduced after UV-B stress (Huang et al., 2002). Almost all of the identified proteins which belonged to the group of protein biosynthesis were down-regulated, except an isoform of Msr-A (spot 106). These spots included three isoforms of Msr-A, which matched its theoretical PI (spots 103, 104, and 105), aminopeptidase P (spot 107), and 30S ribosomal protein S2 (spot 108). The protein of 30S ribosomal protein S2 (30S-RP) was also found to be depressed at the mRNA level under UV-B stress (Huang et al., 2002).

Fig. 4.

The bar graphs display relative synthesis rates (treated to control) of the individual proteins at the different stress time points. These proteins were involved in the long-term UV-B responsive protein network (Fig. 5). Bars 1–4 represent the control and 72, 84, and 96 h of UV-B stress, respectively. Error bars were displayed. M-D, malate dehydrogenase; ASC1, anthranilate synthase component I; CpcG, phycobilisome rod-core linker polypeptide; DMEK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; b-PC, phycocyanin b subunit; DXPS, 1-deoxy-D-xylulose-5-phosphate synthase; PALP, phycocyanin associated linker protein; A-P, aminopeptidase P; MSR-A, methionine sulphoxide reductase A; b-TS, tryptophan synthase subunit beta; C-S, cysteine synthase; 30S-RP, 30S ribosomal protein S2; GAL, glutamate–ammonia ligase; GroEL, chaperonin GroEL; D-L, DNA ligase; PCT, 3-phosphoshikimate1-carboxyvinyltransferase; S-A, sulphate adenylyltransferase; A-O, amine oxidase; HSP90, heat shock protein 90; GrpE, heat shock protein; a-DDRP, DNA-directed RNA polymerase alpha subunit; SOS, SOS function regulatory protein; P-D, phytoene desaturase; T1, transketolase; T2, transaldolase; GST, glutathione S-transferase; P-M, phosphoglycerate mutase; PRK, phosphoribulokinase; M-E, malic enzyme; I-D, isocitrate dehydrogenase; SSADH, succinate-semialdehyde dehydrogenase.

On the other hand, up-regulated proteins were mainly involved in four functional categories: (i) DNA repair, (ii) HSP family, (iii) NADPH generation, and (iv) cellular antioxidative reactions (see Supplementary Table S2 at JXB online; Fig. 4). The identification of increased amounts of the enzymes involved in DNA repair—DNA ligase (spot 044), SOS function regulatory protein (spot 097), and DNA-directed RNA polymerase alpha subunit (spot 110)—suggested that DNA is the main target of UV-B stress in Synechocystis sp. PCC 6803. All proteins belonging to the functional category ‘HSP family’ were up-regulated in response to long-term UV-B stress. These included heat shock protein 90 (spot 048), chaperonin GroEL with two isoforms (spots 049 and 050), and GrpE protein with two isoforms (spots 051 and 052). For most of the induced HSPSIn UV-B conditions, the corresponding gene was also found to be induced by transcriptomics (Huang et al., 2002). The third group of NADPH generation comprised enzymes of succinate-semialdehyde dehydrogenase (spot 055), transketolase (spot 057), transaldolase (spot 058), malic enzyme with two isoforms (spots 060 and 061), and isocitrate dehydrogenase (I-D) with three isoforms (spots 062, 063, and 064). The up-regulation of these enzymes mentioned above, combined with the down-regulation of phosphoglycerate mutase (spot 056), indicated that more NADPH was generated (Kletzien et al., 1994; Busch and Fromm, 1999; Pocsi et al., 2004). As a fourth group, phytoene desaturase (spot 043), amine oxidase (spot 045), and glutathione S-transferase (spot 047) were found among the accumulated proteins after 2-DE separation of proteins from long-term stress in cells of Synechocystis. This group of proteins indicates a response to an impaired redox balance in the cell.

Discussion

The identification of proteins that are differentially expressed after UV-B irradiation in cyanobacteria is an important step in identifying the mechanisms that participate in these responses. To this end, a proteomic analysis was carried out by utilizing Synechocystis under UV-B stress. Comparative proteomics of the cytoplasm of Synechocystis under control conditions and UV-B stress uncovered differences in expression of 112 stress-related proteins. One aim of this work was to identify proteins quantitatively affected by short-term and long-term UV-B radiation. It was surprising to observe that the transient UV-B response has a lower overlap with the later long-term UVB response (see Supplementary Tables S1 and Supplementary Data at JXB online). Furthermore, it is interesting to note that the ratio of up-regulated proteins to down-regulated proteins is larger in long-term response-proteins than in short-term response-proteins. This may be because of the increased time interval; more metabolisms of cyanobacteria are activated by the stress environment to make the cyanobacteria adapt to it. Among the functional group ‘translation’, it should be noted that almost all the short-term response-proteins were up-regulated and all long-term response-proteins were down-regulated in protein biosynthesis (see Supplementary Tables S1 and Supplementary Data at JXB online). Taken together, the regulated expression response patterns of all the proteins readily mirror the fact that the process from acceleration to inhibition of novel protein biosynthesis is required for the survival and growth of Synechocystis sp. PCC 6803 under UV-B stress. An active quality control system of proteins inside cells plays a crucial role in modulating the accommodation of Synechocystis sp. PCC 6803 under UV-B stress.

There is a pre-existing set of knowledge about how cyanobacteria and other algae respond to UV-B stress. However, how cyanobacteria maintain survival and growth under UV-B stress, through intelligent regulation of their metabolic network and defence reactions needs additional research. In this study, the combined results of the proteomic and physiological data presented here for the first time render a coherent image of a long-term UV-B responsive network with most of the 84 long-term response-proteins identified in Synechocystis sp. PCC 6803 (Figs 4, 5; see Supplementary Table S2 at JXB online). This network consists of changes of several functional components, including reduced biosynthesis of amino acids, weakening of protein biosynthesis, impaired photosynthesis, imbalance between ROS production and scavenging, an enhanced cell defence system, and others (Figs 4, 5). These changes of metabolic reactions and redox balance eventually led cyanobacteria to a new homeostasis which can adapt to or/and resist external adverse stresses. Such a protein network allows us to fully understand and describe a possible responsive strategy of cellular activities occurring in UV-B treated Synechocystis sp. PCC 6803.

Fig. 5.

A putative model of the long-term UV-B stress-responses in Synechocystis sp. PCC 6803. Only part of the long-term UV-B responsive proteins are showed, with those up-regulated marked by ↑ and those down-regulated marked by ↓. OX, oxidation; RED, reduction; DMEK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; b-PC, phycocyanin b subunit; DXPS, 1-deoxy-D-xylulose-5-phosphate synthase; PALP, phycocyanin associated linker protein; A-P, aminopeptidase P; MSR-A, methionine sulphoxide reductase A; b-TS, tryptophan synthase subunit beta; C-S, cysteine synthase; 30S-RP, 30S ribosomal protein S2; GAL, glutamate–ammonia ligase; GroEL, chaperonin GroEL; D-L, DNA ligase; PCT, 3-phosphoshikimate1-carboxyvinyltransferase; S-A, sulphate adenylyltransferase; A-O, amine oxidase; HSP90, heat shock protein 90; GrpE, heat shock protein; a-DDRP, DNA-directed RNA polymerase alpha subunit; SOS, SOS function regulatory protein; P-D, phytoene desaturase; T1, transketolase; T2, transaldolase; GST, glutathione S-transferase; P-M, phosphoglycerate mutase; PRK, phosphoribulokinase; M-E, malic enzyme; I-D, isocitrate dehydrogenase; SSADH, succinate-semialdehyde dehydrogenase. (This figure is available in colour at JXB online.)

Current research shows that when Synechocystis sp. PCC 6803 is exposed to UV-B, nucleic acid is damaged. It also yields an excessive ROS formation by photosynthetic pigments and redox components. These pigments and components include chlorophylls, phycobiliproteins, and quinones, all of which exhibit absorption in the UV range. The excessive ROS levels in Synechocystis sp. PCC 6803 cause an imbalance of the original redox homeostasis which tends to exhibit an elevated oxidative intensity. This would lead to changes of biomolecular metabolism (Stork et al., 2005).

It has been reported that a large part of the UV-B energy was directly absorbed by phycobiliproteins and chlorophyll proteins (Lao and Glazer, 1996). These pigmented complexes represent the main target of UV absorption. Phycobilins in cyanobacteria can act as photosensitizers and produce ROS under UV or visible light excess by the reaction of chromophore triplet states with molecular oxygen (Zolla and Rinalducci, 2002). The excited chromophore itself contributes to a large extent to the observed damage. As a result, the elevated ROS levels inhibit the expression of light-harvesting antenna proteins such as b-PC and phycocyanin associated linker protein and the down-regulation of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase and 1-deoxy-D-xylulose-5-phosphate synthase has a negative influence on the biosynthesis of carotenoids and chlorophylls. This led to the reduced light-harvesting capability of Synechocystis sp. PCC 6803. Furthermore, phosphoribulokinase which is the key enzyme involved in the Calvin cycle declined under oxidative stress. All of these eventually impaired photosynthesis (Fig. 1B–D).

Synthesis and degradation of biomolecules (particularly proteins) are the most important links in the life processes. Under the long-term UV-B stress, the expression of three isoform of Msr-A, aminopeptidase P, and 30S-RP which were involved in protein biosynthesis were down-regulated. Two major roles of Msr have been proposed. First, Msr activity can repair a few key proteins that then maintain the function of other proteins, including housekeeping ones (Ezraty et al., 2004). Second, Msr-dependent reduction of some Met-containing proteins allows such proteins to serve as ROS quenchers or sinks by a continual oxidation–reduction cycle on the surface of methionine residues. Such a cycle is thought to be especially important under stress conditions (Stadtman et al., 2003). It is not surprising that UV-B damaged the equilibrium of protein metabolism between biosynthesis and degradation. This also exhibited functional characteristics which skew into a speeding up of protein degradation and a slowing down of protein biosynthesis for long-term stress (Figs 4, 5).

In the stress stage, proteins associated with amino acid biosynthesis were also depressed. Amino acid is an essential component of protein biosynthesis and a crucial factor in determining body growth and vigour. Moreover, amino acid can influence metabolism and resistance to stress. In general, the down-regulated enzymes of amino acid biosynthesis would have harmful effects on many aspects of life. Figure 1 showed that after 1 W m⁻² UV-B radiation, the growth rate of Synechocystis sp. PCC 6803 was retarded. The reduction in amino acid biosynthesis is one of the key reasons for this phenomenon.

On the other hand, the increased ROS level might lead to activation of many different stress-defence pathways. As mentioned above, the photosystem was damaged by UV-B. Following the weakening of the Calvin cycle, the pentose phosphate pathway (PPP) became enhanced. The up-regulation of transaldolase and transketolase and the down-regulation of phosphoglycerate mutase supported this conclusion. Transaldolase and transketolase are two key enzymes in the non-oxidative steps of PPP. It is known that the Calvin cycle and the PPP perform most of the carbohydrate metabolism in cyanobacteria since the Krebs cycle is incomplete in cyanobacteria. The expression levels of transaldolase and transketolase increased 2.1-fold and 3.1-fold, respectively, when the photosystem was damaged by UV-B. This refers to an enhanced PPP particularly when the Calvin cycle, where these enzymes also have a role in the reductive biosynthetic process, is scaled down. The PPP is the main NADPH-producing pathway. At the same time, the expression of IDPc, SSADH, and malic enzyme increased. Together, these changes are expected to favour the production of NADPH which is a well-known molecule with great importance in both GSH recycling and the thioredoxin/glutathione redox cycle against ROS (Figs 4, 5). There are a considerable number of NADPH-consuming stePSIn plant defence reactions which are readily found in the literature.

Many proteins with an antioxidative, a DNA repair, or a protein repair function such as GST, amine oxidase, phytoene desaturase, DNA ligase, SOS function regulatory protein, RNA polymerase, and HSPS increased their expression level after UV-B exposure (Figs 4, 5; see Supplementary Table S2 at JXB online). This led to the enhancement of the cellular defence level. Such defence systems play an important role in maintaining the survival and growth of Synechocystis sp. PCC 6803 under strong and sustained UV-B stress (Fig. 5).

The activated antioxidative systems in cells of cyanobacteria possess a strong capability for removing ROS, which can reduce the intracellular ROS levels and attenuate the oxidative damage, thus ultimately establishing a new redox homeostasis. The function of amine oxidase is to oxidize pyridoxine 5′-phosphate (PNP) and pyridoxamine 5′-phosphate (PMP) into pyridoxal 5′-phosphate (PLP), which shows quenching activity toward singlet oxygen and superoxide. Moreover, it has the highest antioxidant activity among vitamin B6 compounds (Bilski et al., 2000; Ohta and Foote, 2002; Chumnantana et al., 2005). Glutathione S-transferases (GSTs) are a group of multifunctional proteins involved in the detoxification of a wide spectrum of compounds. GSTs with glutathione (GSH) peroxidase activity, participate in the detoxification of products created by oxidative damage and thereby protect cells against such stress (Dixon et al., 1998). This protection mechanism is often accelerated by increased amounts of GSH (May et al., 1998). Molecular genetic analyses show that the over-expression of the UV inducible GST could increase the tolerance of the transgenic plants to UV radiation (Liu and Li, 2002).

The DNA repairing system can effectively protect against UV-induced damage. The DNA ligase is known to be important for the DNA repair pathway. Its up-regulation may be in response to relatively harsh DNA damaging conditions of UV-B stress (Montecucco et al., 1992). SOS function as regulatory proteins expressed when the cells are exposed to agents that damage DNA or block replication (e.g. UV irradiation). They have been shown to improve cellular survival by providing increased repair capacity and by transiently retarding cell division (Walker, 1984; Shinagawa, 1996). The RNA polymerase transcription machinery acts as a molecular motor that traverses large parts of the genome on a regular basis. It has been suggested that the transcription machinery may play an important role in sensing DNA damage and activating DNA repair and stress response pathways (Ljungman, 2007).

Most HSPs have strong cytoprotective effects. These maintain proteins in their functional conformations, preventing aggregation of non-native proteins. They also maintain a refolding of denatured proteins to regain their functional conformation and removal of non-functional but potentially harmful polypeptides (arising from misfolding, denaturation or aggregation). This often occurs in an ATP-driven process, and is therefore frequently up-regulated under stress conditions in many organisms (Slabas et al., 2006; Suzuki et al., 2006; Timperio et al., 2008). The stronger heat shock responses could reflect greater damage caused by UV-B illumination.

Exposure of Synechocystis cells to UV-B causes major changes in both proteomic and transcriptomic patterns. Huang et al. (2002) investigated the Synechocystis sp. strain PCC 6803 response to UV-B using microarray-based global gene expression approaches. They revealed that 21 genes exhibited significantly elevated mRNA levels and 40 genes exhibit significantly reduced mRNA levels after 2 h exposure. Comparative analysis of our proteomics data with their transcriptomics data displays only 39 protein spots sharing identical directed fold alteration at both mRNA and protein level among the 112 identified proteins (see Supplementary Tables S1 and Supplementary Data at JXB online). Several reasons might account for the low mRNA–protein correlation. One possible explanation is the difference in UV-B irradiation dose between the two independent groups of experiments. In Huang's study, a short-term (20 min or 2 h) response of Synechocystis to UV-B at transcription level was explored, while in our research, the proteomic level was investigated after a longer (8 h or 3–4 d) exposure to UV-B. The non-overlapping time-course might yield different results of fold change even in the same experimental system. Another key aspect is due to differential half-lives of transcripts and proteins or translation-on-demand (Schmidt et al., 2007). In our research, many proteins which can help cyanobacteria adapt to or/and resist external UV-B stresses were not mentioned in the transcriptomic research (Huang et al., 2002). For example, proteins involved in amino acid biosynthesis, NADPH generation, and cellular antioxidative reactions (Fig. 4; see Supplementary Tables S1 and Supplementary Data at JXB online) were not mentioned. This research is complementary to previously carried-out transcriptomic analysis. However, the mechanisms leading to changed protein amounts, which are obviously not directly based on changed transcriptional rate, remain to be elucidated.

Conclusions

In summary, the protein profiling and expression data presented here provide an expanded view of the cytoplasmic proteins expressed and involved in the response of Synechocystis sp. PCC 6803 under short-term and long-term UV-B exposure. Analysis of approximately 1000 protein spots on each two-dimensional electrophoresis gel revealed128 differentially expressed proteins. Among them, 112 protein spots were successfully identified by mass spectrometry to match 75 diverse protein species. These proteins not only include many well-known UV-B induced proteins such as HSP90, GST, chaperonin GroEL, 50S ribosomal protein, 30S ribosomal protein, elongation factor EF, and glutamate–ammonia ligase, but also many novel responsive proteins which have not been found in current research related to UV-B stress (Casati et al., 2005; Xu et al., 2008). Identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in the acclimation of cyanobacteria under UV-B stress.

Combining our proteomic and physiological data with previously published results, a long-term UV-B responsive network was constituted with most of the 84 responsive proteins identified in Synechocystis sp. PCC 6803 under long-term UV-B exposure (Fig. 5). Such a protein network gives us a global view and provides new insights into understanding the possible responsive strategy of cellular activities occurring in the UV-B-treated Synechocystis sp. PCC 6803.

This work was supported by NSFC project 30670476, the NSFC Guangdong Joint project U0633009, the National High Technology Research and Development Program of China (863 Program) 2007AA05Z400, and the MOST overseas co-operation project 20070574. The authors thank Yuan Li, Yunluan Cui, Xiaobo Li, Yan Zhai, and Disi Wang for their invaluable assistance. The authors also thank Professor Jinyuan Liu, Dr Xiangyuan Wan, and Dr Saleem B (Tsingahua University, China) for excellent insights.