Journal of Photochemistry and Photobiology B: Biology 130 (2014) 318–326

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

The cry-DASH cryptochrome encoded by the sll1629 gene in the cyanobacterium Synechocystis PCC 6803 is required for Photosystem II repair István-Zoltán Vass a, Péter B. Kós a, Jana Knoppová b, Josef Komenda c, Imre Vass a,⇑ a b c

Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, P. O. Box 521, H-6701 Szeged, Hungary Faculty of Science, University of South Bohemia, Branisovska 31, Ceske Budejovice, Czech Republic Institute of Microbiology, Academy of Sciences, Opatovicky Mlyn, 379 81 Trebon, Czech Republic

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Article history: Received 26 August 2013 Received in revised form 26 November 2013 Accepted 9 December 2013 Available online 16 December 2013 Keywords: UV-B radiation Photosystem II repair Photolyase Chryptochrome Synechocystis PCC 6803

a b s t r a c t The role of the Syn-CRY cryptochrome from the cyanobacterium Synechocystis sp. PCC 6803 has been a subject of research for more than a decade. Recently we have shown that photolyase, showing strong homology with Syn-CRY is required for Photosystem II repair by preventing accumulation of DNA lesions under UV-B (Vass et al. 2013). Here we investigated if Syn-CRY is also involved in PSII repair, either via removal of DNA lesions or other mechanism? The Dsll1629 mutant lacking Syn-CRY lost faster the PSII activity and D1 protein during UV-B or PAR than the WT. However, no detectable damages in the genomic DNA were observed. The transcript levels of the UV-B and light stress indicator gene psbA3, encoding D1, are comparable in the two strains showing that Dsll1629 cells are not defective at the transcriptional level. Nevertheless 2D protein analysis in combination with mass spectrometry showed a decreased accumulation of several, mostly cytoplasmic, proteins including PilA1 and bicarbonate transporter SbtA. Dsll1629 cells exposed to high light also showed a limitation in de novo assembly of PSII. It is concluded that Syn-CRY is required for efficient restoration of Photosystem II activity following UV-B and PAR induced photodamage. This effect is not caused by retardation of DNA repair, instead the synthesis of new D1 (and D2) subunit(s) and/or the assembly of the Photosystem II reaction center complex is likely affected due to the lack of intracellular CO2, or via a so far unidentified pathway that possibly includes the PilA1 protein. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Cryptochromes (CRYs) are flavin-containing blue light photoreceptors related to photolyases. However, while photolyases are light-dependent DNA repair enzymes [1–4] cryptochromes, although show high sequence similarity to photolyases, seem to lack conventional DNA repair ability [5,6] and are mainly active as signaling molecules. Plant CRYs primarily mediate blue-light stimulation, which constitutes an input pathway for the circadian clock [7]. Certain animal CRYs may also act as photoreceptors [8], while others function as light-independent transcription repressors [9]. Recently a new group of cryptochromes has been identified [10], its homologues were found in diverse organisms (Drosophila, Arabidopsis, Synechocystis, Homo) and denoted as cry-DASH. The most prominent member and initiator of the new group is the Synechocystis cry-DASH (Syn-CRY, encoded by the sll1629 gene), which was the first cryptochrome to be identified from bacteria [10,11]. Since its description as a possible cryptochrome several attempts have been made to associate function to the Syn-CRY protein, but ⇑ Corresponding author. Tel.: +36 62 599 700; fax: +36 62 433 434. E-mail address: [email protected] (I. Vass). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.12.006

its role has not yet been clearly established. Along with the other members of cry-DASH, Syn-CRY has been hypothesized to act as a weak photolyase [10,12–14]. It binds a FAD cofactor [11], which is necessary for photolyases to specifically bind to cyclobutane pyrimidine dimers (CPDs) [15]. However, based on missing amino acid (AA) binding elements [10,16,17] and on absorption spectrum data [11], Syn-CRY probably cannot bind a secondary chromophore, which is present in photolyases [18]. Nevertheless, emphasizing their other presumed function as a photoreceptor, recent studies suggested that Cry-DASH proteins, mainly speaking about Arabidopsis and Synechocystis Cry-DASH, are able to bind secondary pterin chromophores [19–22]. In an initial study, the presence of Syn-CRY in photolyase lacking cells induced a weak CPD photorepair activity in vivo, but showed no photorepair activity in vitro [11]. In addition, it has also been suggested that Cry-DASH proteins from bacterial, plant, and animal sources actually are photolyases, which act on CPDs in single-stranded DNA [13]. Therefore, the role of Cry-DASH proteins in DNA repair is still unresolved. On the other hand, the Syn-CRY protein has been shown to participate in sensing UV-A light and involved in UV-induced phototaxis of cyanobacteria [22]. A further role assigned to cry-DASH in cyanobacterial systems is the

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regulation of the production of microsporine like amino acids (MAAs), which act as UV screening pigments [23]. While light provides energy to the biosphere via photosynthetic energy conversion, as well as input signals for environmental acclimation of photosynthetic and non-photosynthetic organisms via photoreceptors, at the same time light can cause damage to biological systems. This damaging effect is of special importance for photosynthetic organisms, which are unavoidably exposed to incident sunlight. The most detrimental component of sunlight that reaches the Earth’s surface is the UV-B (280–315 nm) spectral range, which can damage DNA, proteins and lipids, and other cell components. In photosynthetic organisms DNA is partially protected against UV-B light by absorption of pigmented complexes [24], while the photosynthetic apparatus, especially the Photosystem II (PSII) complex is an important UV-B target (see Refs. [25,26]). Within PSII the primary action site of UV-B radiation is the catalytic manganese cluster of the water oxidizing complex, whose damage leads to inhibition of light induced electron transport [27–30]. The D1 and D2 protein subunits, which form the backbone of the reaction center complex are also damaged by UV-B exposure, which leads to their degradation [31–35]. UV-B induced damage of PSII can be restored via repairing the PSII core complex, which proceeds via: (i) Proteolytic removal of the damaged D1 and D2 protein subunits. (ii) Transcription of mRNA from the psbA and psbD genes encoding the D1 and D2 subunits, respectively. (iii) Production of new protein subunits from the respective mRNA pools. (iv) Incorporation of the newly synthesized protein copies into the PSII complex, and finally (v) religation of redox cofactors and reactivation of PSII (for reviews see [36,37]). In a recent work we have shown that the PSII repair cycle is connected to the photolyase dependent repair of UV-B damaged DNA [38]. In the cyanobacterium Synechocystis 6803 the absence of the photolyase enzyme, encoded by the phrA (slr0854) gene, leads to accumulation of damaged DNA, which induces the retardation of psbA gene transcription and decrease of D1 protein amount in parallel with the accelerated loss of PSII activity. It was concluded that unrepaired DNA damage interrupts the PSII repair cycle at the step of gene transcription, and thus inhibits de novo D1 (and D2) protein synthesis [38]. Based on the high sequence homology of Syn-CRY (phrB) with photolyase (phrA) and on the weak in vivo CPD photorepair activity of Synechocystis cells, which lack the photolyase but contain the Syn-CRY protein [11], we intended to clarify if Syn-CRY could be involved in PSII repair. Our data show that a Synechocystis mutant which lacks the phrB (sll1629) gene indeed shows retarded PSII repair and decreased D1 protein amount as would be expected from a limited DNA repair activity. However, this mutant does not accumulate unrepaired CPDs or other DNA products (such as 8-oxoG produced by oxidative damage of DNA) and is not affected in psbA gene transcription either. On the other hand, the lack of the phrB gene product leads to the decreased level of bicarbonate transporter SbtA and a PilA type protein, which has been shown to participate in Syn-CRY mediated phototaxis [22,39], and has been reported to participate in Chl delivery to the PSII complex, as well as to the antennae [40]. The data are discussed in terms of an indirect role of the Syn-CRY protein in PSII repair, which is mediated via the regulation of levels of CO2/bicarbonate transporters and/ or PilA1 protein production.

2. Materials and methods 2.1. Cell cultures and light treatment conditions Synechocystis PCC6803 (denoted as Synechocystis hereafter) wild type and its Syn-CRY lacking mutant (Dsll1629 mutant, a kind gift

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of Prof. Alfred Batschauer), which was constructed by interrupting the gene with a kanamycin cassette [41], were cultured in an incubator with an orbital shaker (120 rpm) in BG-11 medium. Within the incubator 30 °C temperature, 3% CO2 enriched atmosphere, and 40 lmol photons m 2 s 1 PAR was maintained. Cells were harvested in their exponential growth phase by centrifugation (6500 g, 5 min, 24 °C). Pellets were resuspended in fresh BG-11 medium at 6.5 Chl lg mL 1 and then incubated for one hour before experiments. The UV-B illumination source was a Vilber Lourmat VL-215 M lamp on which cellulose acetate filter (Clarfoil 0.1 mm, cut off at 290 nm) was applied to screen out any possible UV-C radiation. The emitted radiation has peak intensity at 312 nm, and has about 35 nm half-bandwidth. UV-B treatments were carried out at 30 °C. Cell cultures formed a 2.5 cm high layer, while being continuously stirred in square glass containers. The intensity of UV-B radiation was 3.0 W m 2 (8.0 lmol photons m 2 s 1) at the surface of the cell suspension as measured with a Cole-Parmer radiometer (model 97503-00) equipped with a 312 nm sensor. This value was significantly less within the cell suspension, approximately 0.3 W m 2 (0.8 lmol photons m 2 s 1), as calculated by taking into account the absorption by the optically dense sample according to [42]. Prior to the onset of UV-B treatments cells were kept under 50 lmol photons m 2 s 1 Low intensity white Light (LL) for 60 min. These conditions were also used as background illumination during the UV-B treatments, as well as during the recovery period following the UV-B exposure. UV-B radiation was applied either in the absence or in the presence of the protein synthesis inhibitor lincomycin (300 lg/mL) in order to block protein synthesis. For High Light (HL) treatment cells were exposed to 500 lmol photons m 2 s 1 of white light illumination either in the absence or in the presence of the protein synthesis inhibitor lincomycin (300 lg mL). White light and UV-B intensities were measured using a LiCor Photometer (DMP Ltd.) equipped with a PAR sensor and a Cole Parmer Radiometer equipped with a UV-B (312 nm) sensor, respectively. 2.2. Radioactive labeling of proteins and preparations of thylakoid membranes Radioactive pulse labeling of the cells was performed using a mixture of [35S]Met and [35S]Cys (Trans-label, MP Biochemicals, Irvine, USA) as described previously by Komenda et al. [55]. Cells (75 lg of Chl) were exposed to irradiance of 500 lmol photons m 2 s 1 of white light at 29 °C for 20 min and afterwards were frozen in liquid nitrogen and used for isolation of thylakoids. To prepare cyanobacterial thylakoid membranes harvested cells were washed, resuspended in the thylakoid buffer containing 25 mM MES/NaOH, pH 6.5, 5 mM CaCl2, 10 mM MgCl2, and 20% glycerol and broken using glass beads. The broken cells were pelleted (20,000g, 15 min) and resuspended again in the thylakoid buffer to obtain the membrane fraction. 2.3. Electrophoresis and immunoblotting Analysis of membrane proteins under native conditions was performed by a clear native (CN)-PAGE as described in Wittig and Schägger [43]. The isolated thylakoid membranes were solubilized in 1% n-dodecyl-b-maltoside and analyzed at 4 °C in a 4–14% polyacrylamide gel. The gel was photographed, scanned for Chl fluorescence using LAS 3000 scanner (Fuji) and cut into slices containing sample lanes. After incubation for 30 min in 25 mM Tris/ HCl, pH 7.5 containing 1% SDS (w/v) and 1% dithiothreitol two slices were placed on the top of single 12–20% linear gradient polyacrylamide gel containing 7 M urea [44] and individual proteins of

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thylakoid membrane complexes were resolved in the second dimension. Gel was scanned by LAS3000 (Fuji), dried, exposed to a Phosphorimager plate, which was scanned by Storm (GE Healthcare, Vienna, Austria). For identification of selected protein, stained bands from 2D gels were excised and mass spectra were measured on a APEX-Qe 9.4T FT-MS instrument equipped with a Combi ESI/ MALDI ion source (Bruker Daltonics, Billerica, MA) as described in [45]. 2.4. Assessment of photosynthetic activity PSII activity was assessed, during and after UV-B or HL treatments and recovery periods, via measuring changes in the yield of variable Chl fluorescence, which reflects the relative amount of functional PSII complexes. Variable fluorescence was calculated from the initial amplitudes of flash induced fluorescence signals (Fv = Fm Fo) after different periods of UV-B or HL treatments, and shown relative to the untreated samples. These measurements were carried out with an FL 3000 Fluorometer (Photon Systems Instruments Ltd.), using 1 mL samples in the absence and presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Repetitions were performed on 3 biologically independent samples at preset time points (see Fig. 1). The data were visualized and evaluated using the Fluorwin software, version 3.6.3.11 and Origin.

adding an equal amount of phenol containing (5%, v/v) ethanol. After collecting the cells (10,000g, 10 min, 4 °C) from this solution, we applied the hot-phenol method to isolate total RNA [48]. The resulting, rehydrated RNA samples underwent a DNase treatment (Turbo DNA-Free Kit, Ambion), which was followed by a reverse transcription (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). We used the synthesized cDNA as template in qPCR reactions. CT values of psbA3 cDNA were normalized to CT values of rnpB (subunit of RNase B), the latter being constitutively expressed. The values on the graphs were further normalized to the values of samples (t = 0). 2.7. Detection of UV-B induced DNA damage by alkaline gel method

Thylakoid membranes were isolated from samples taken after 0, 120 and 270 min UV-B or HL treatments, as well as after the subsequent 90 min of WL recovery. The isolation procedure was described in [46]. Thylakoid samples, each containing 1 lg of Chl, were separated by SDS-PAGE, on a 12% gel, blotted on nitrocellulose membrane and visualized with specific antibodies according to [35].

gDNA from UV-B stressed WT and Dsll1629 cells was isolated as described in [38]. Detection of cyclobutane pyrimidine dimers (CPDs), the predominant UV-B-induced DNA lesion, was carried out by incubating gDNA with T4 Endonuclease V. This enzyme specifically cleaves one strand of the DNA molecule at pyrimidine dimer sites, causing single strand brakes [49–51]. The digested samples were separated on a 0.6% alkaline agarose gel, previously soaked in alkaline electrophoresis buffer (50 mM NaOH, 1 mM EDTA). The same solution was used as running buffer. For the detection of 7,8-dihydro-8-oxoguanine (8-oxoG) lesions [52] gDNA was incubated with Formamidopyrimidine-DNA glycosylase (FPG). FPG is a DNA repair enzyme that excises oxidized purines from damaged DNA with a preference for 8-oxoGs, thus cleaving the DNA’s backbone [53]. The presence of unrepaired pyrimidine dimers or 8-oxoGs, in the alkaline environment yields small, single stranded DNA fragments, which show up as a smear on the alkaline gel. The relative decrease of the apparent average length of the resulted fragments is representative of the encountered direct UV-B damages. Nucleic acids were visualized using ethidium–bromide.

2.6. Analysis of psbA3 gene expression

2.8. Statistical analysis

Expression level of the psbA3 gene was assessed by qPCR as described earlier [47]. Briefly, we harvested 10 ml of cell suspension at any given time point during experiments, and fixed the cells by

Variability of data points was evaluated with one-way ANOVA, at P = 0.05.

2.5. D1 protein analysis

3. Results 3.1. Absorption and fluorescence characteristics Measurements of room temperature absorption and 77 K fluorescence spectra showed the same cellular content of chlorophyll (Chl) and photosystems in both strains the WT and the mutant strain that lacks the sll1629 gene. The Dsll1629 mutation only caused a slightly increased level of phycobilisomes (peak at 620 nm) (Supp. Fig. 1). 3.2. DNA repair ability of Syn-CRY lacking cells

Fig. 1. UV-B-induced DNA damage assessment via alkaline gel separation of gDNA from Synechocystis cells. gDNA isolated from UV-B-treated Dsll1629 and DphrA (Dslr0854, photolyase lacking) cells was digested with T4 Endonuclease V or Formamidopyrimidine-DNA glycosylase (FPG) and separated on alkaline gels. UV-B treatment for Panels A, B, C: 270 min of 8 lmol photons m 2 s 1 UV-B + 50 lmol photons m 2 s 1 white light; UV-B treatment for Panel D: 20 min of 20 lmol photons m 2 s 1 UV-B. The gels were neutralized and soaked in ethidium-bromide for visualization. Panel A: gDNA from Dsll1629 cells digested with T4 Endonuclease V. Panel B: gDNA from DphrA cells digested with T4 Endonuclease V. Panel C: gDNA from Dsll1629 cells digested with FPG. Panel D: gDNA from DphrA cells digested with FPG.

In an attempt to address the potential involvement of Syn-CRY in repairing DNA, damaged by UV-B or oxidative stress, we isolated gDNA from Synechocystis samples after 270 min of 8 lmol photons m 2 s 1 UV-B exposure in the presence of 50 lmol photons m 2 s 1 background LL. DNA repair assessment was carried out by trying to detect the predominant UV-B-induced DNA lesions, cyclobutane pyrimidine dimers (CPD) on these gDNA samples. The isolated gDNA samples were treated with T4 Endonuclease V, an enzyme that specifically cleaves the DNA strands only at pyrimidine dimer sites, producing single strand brakes. The treated gDNA samples were then separated on alkaline gels that resulted in single stranded DNA fragments if CPD lesions were present on

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the DNA strands. The more unrepaired CPDs were present on the DNA strands, the shorter the fragments are expected to be present on the gels, causing a smear of the DNA band. We found that after 270 min of UV-B treatment there was no detectable amount of unrepaired CPD lesions on the DNA strands of Dsll1629 cells (Fig. 1, panel A). In contrast, in cells lacking the DNA photorepair enzyme photolyase (DphrA), the homolog protein of Syn-CRY, CPDs accumulate under the same stress conditions as was shown earlier [38]. Applying the same procedure to gDNA, isolated from UV-B-irradiated DphrA cells, smears are formed on the alkaline gel from the resulting small DNA fragments (Fig. 1, panel B). We also tested the presence of unspecific DNA lesions on the DNA strands with a qPCR method, as described in [38] with similar results showing no significant amount of accumulated lesions (not shown). Other types of specific DNA lesions that are often produced under stress conditions are oxidation related lesions, of which the most frequent ones are the oxidized purines, such as 7,8-dihydro-8-oxoguanine (8-oxoG). We also checked if Syn-CRY could be involved in the elimination of 8-oxoGs by digesting gDNA from UV-B stressed Dsll1629 cells with Formamidopyrimidine-DNA glycosylase (FPG). This enzyme cleaves the backbone of the DNA and excises oxidized purines, with a preference for 8-oxoGs. If lesions are present, the enzyme induces fragmentation of the respective DNA strand and under alkaline conditions, a smear will appear on the gel (Fig. 1, Panel D). Our results, however, indicate that no detectable amounts of 8-oxoGs accumulate during UV-B stress in cells lacking Syn-CRY (Fig. 1, panel C). These data together with previous literature results show that the Syn-CRY protein is unlikely to be involved in the elimination of DNA lesions, and suggest a function of Syn-CRY in other processes than DNA repair. 3.3. Changes in PSII activity induced by UV-B and visible light (HL) stress in WT and Dsll1629 cells We aimed to investigate the possible role of Syn-CRY (sll1629) in light induced damage and subsequent restoration of PSII activity. In order to achieve this aim changes in the amount of functional PSII, as quantified by flash induced Chl fluorescence measurements, were compared in WT Synechocystis and its SynCRY deficient mutant (Dsll1629) after exposure to either UV-B or visible light (HL) stress. The UV-B stress experiments lasted for 360 min, and consisted of 2 phases of illumination (Fig. 2A). In the first phase cells were exposed to 8 lmol photons m 2 s 1 UV-B in the presence of 50 lmol photons m 2 s 1 background LL for 270 min. During this phase the Dsll1629 cells, which are unable to produce Syn-CRY, suffered significantly larger losses in their PSII activity than the WT cells. After only 30 min following the onset of UV-B radiation PSII activity in Dsll1629 cells decreased to almost half of its initial value, while WT cells were able to maintain their activity at around 80% (Fig. 2A open circles and squares), which represents a significant difference (F = 0.013, at P = 0.05) between the two cell lines. This large initial difference, amounting to 25% in average between WT and Dsll1629 cells, was maintained throughout the course of the UV-B illumination, only being slightly reduced at the very end of the first phase. The second phase of the experiment provided a recovery period for the cells in which UV-B radiation was absent, but the 50 lmol photons m 2 s 1 background LL was retained for 90 min, which helped the cells to partially recover their PSII activity. However, the initial UV-B stress induced difference persisted during this recovery phase as well. PSII activity in WT cells reached close to 70% of its pre-stressed value at the end of recovery phase from the 60% value at the end of the UV-B treatment, while PSII activity in the Dsll1629 cells rose from around 40% to only 45% of their initial values.

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The maintenance of PSII activity under UV-B stress requires efficient protein synthesis. When protein synthesis was inhibited by the addition of lincomycin PSII activity decreased at the same rate in both strains, reaching as low as 20% in only 80 min under exposure to 8 lmol photons m 2 s 1 UV-B and 50 lmol photons m 2 s 1 background LL (Fig. 2A closed circles and squares). In the presence of lincomycin the loss of activity reflects the efficiency of PSII photodamage in itself without the influence of protein synthesis dependent repair. Therefore, the parallel loss of PSII activity in WT and Dsll1629 cells in the presence of lincomycin demonstrates that the rate of UV-B induced photodamage is not affected by the lack of the Syn-Cry. On the other hand the difference between PSII activities in the absence and presence of lincomycin reflects the amount of PSII centers which are repaired or replaced during the UV-B treatment. Since this difference is clearly (F = 0.043, at P = 0.05) smaller in the Dsll1629 cells than in the WT it can be concluded that the efficiency of PSII repair/replacement is decreased in cells which are unable to produce the SynCRY protein. In order to clarify if retardation of PSII repair by the Syn-CRY deletion is specific to UV-B light, or represents a more general phenomenon the effect was also tested under strong illumination by visible light. As shown in Fig. 2B exposure to 500 lmol photons m 2 s 1 (HL) in the presence of lincomycin induced a parallel loss of PSII activity in the WT and Dsll1629 mutant cells. However, in the absence of lincomycin when protein synthesis is functional the light induced loss of PSII activity was more pronounced in the Syn-CRY lacking mutant than in the WT (Fig. 2B). Similar to the effect of UV-B irradiation, a significant difference (F = 0.045, at P = 0.05) developed in PSII activity between WT and Dsll1629 cells, after only 30 min of HL irradiation, which was maintained during the rest of the experiment. In addition, restoration of PSII activity following the HL exposure is also retarded in the absence of the Syn-CRY protein. These findings were confirmed by oxygen evolution measurements both in case of UV-B and HL stress (not shown).

3.4. D1 protein level changes by UV-B and visible light stress in WT and Dsll1629 cells One of the primary targets of UV-B radiation and a key component of PSII repair is the D1 subunit of the PSII reaction center [31,34,35]. Light induced degradation and subsequent de novo synthesis of the D1 protein occurs also under HL stress (for reviews see [36,37]). During our experiments the D1 protein level remained almost unaffected in the WT cells during the UV-B treatment, which was performed in the presence of 50 lmol photons m 2 s 1 background LL that promotes D1 turnover [54] (Fig. 3A). In contrast, the Dsll1629 cells apparently lost a portion of their D1 protein pool by the end of the UV-B treatment. A significant difference in D1 protein levels appeared between WT and Dsll1629 cells after 270 min of UV-B irradiation (F = 0.001, at P = 0.05) and the D1 protein loss was only partly restored during the recovery period under background WL illumination following the UV-B exposure. Similar results were observed when cells were exposed to HL (500 lmol photons m 2 s 1) conditions The loss of the D1 protein amount was clearly larger in the Dsll1629 mutant cells than in the WT after 90 min of HL (F = 0.014, at P = 0.05) and only a slight restoration of the D1 protein content could be observed during recovery after the HL treatment, when only LL exposure is present (Fig. 3B). The inability of the mutant cells to maintain their D1 level under the UV-B or HL stress concurs with the observed loss of PSII activity shown in Fig. 2A and B and supports the idea that absence of the Syn-CRY protein leads to inefficient PSII repair/replacement under UV-B, or HL stress conditions.

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Relative fluorescence amplitude (%)

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Fig. 2. UV-B and HL-induced loss of PSII activity in Synechocystis cells. (A) Wild type and Dsll1629 mutant cells were exposed to 8 lmol photons m 2s 1 UV-B illumination, in addition to 50 lmol photons m 2 s 1 low intensity background white light (LL) for 270 min, either in the presence (closed symbols) or in the absence (open symbols) of protein synthesis inhibitor lincomycin (Linc). This initial stress-treatment was followed by administration of 90 min of 50 lmol photons m 2 s 1 LL, as means of aiding the recovery of cells from the UV-B stress. (B) Wild type and Dsll1629 cells were both treated with 500 lmol photons m 2 s 1 of high intensity white light (HL) for 90 min, after which 50 lmol photons m 2 s 1 of LL was applied for another 90 min. The data represent means obtained from 3 independent experiments with the indicated standard deviations.

Fig. 3. Changes in D1 protein amount under UV-B and HL stress in thylakoid membranes of Synechocystis cells. (A) Wild type and Dsll1629 mutant cells underwent 270 min of 8 lmol photons m 2 s 1 UV-B stress, with a background illumination of 50 lmol photons m 2 s 1 low intensity white light (LL). An additional 90 min of 50 lmol photons m 2 s 1 LL was applied to the cells to check for their ability to mitigate the initial stress symptoms. (B) Both cell lines were exposed to 500 lmol photons m 2 s 1 of high intensity white light (HL) for 90 min, followed by a 90 min recovery period under 50 lmol photons m 2 s 1 of LL. Thylakoid samples were isolated from cells taken during the stress and recovery phases of the experiments and separated by SDS-PAGE. D1 proteins were detected and visualized by immunoblotting. Loaded samples contained 50 ng of Chl, which corresponds to 5% on the concentration series. The bar diagrams show the densities of the D1 bands obtained from 3 independent experiments with the indicated standard error.

deletion on accumulation and synthesis of subunits of photosynthetic complexes and other thylakoid membrane proteins. For assessment of the content of oligomeric forms of PSII we also used detection of Chl fluorescence directly in the native gel (Fig. 4, 1D fluor) which showed a decrease in the amount of the dimeric PSII core complex (RCC(2)) in the mutant. This finding agreed with the intensity change of large PSII subunits (CP47, CP43, D1, D2) resolved in the second dimension. The Syn-CRY mutant also contained a higher amount of unassembled CP47 (seen as two spots on 2D gels which are designated as 1) both in the stained gel and in the autoradiogram, on the other hand the level of unassembled CP43 was very similar in both strains (Fig. 4, circles). In both strains we also observed labeled bands of incompletely processed form of the D1 protein (iD1) present in the PSII reaction center (RCII) assembly complexes lacking CP43 and CP47 antennae [55], but the amount of this form was largely diminished in the mutant. This suggests, together with the increased level of free CP47, that the mutant suffers from deficiency in the de novo assembly of RCII complex and this could be related to its inability to efficiently cope with increased intensity of visible or UV-B light. The 2D gel also revealed modified accumulation and synthesis of several PSII-unrelated proteins in the Syn-CRY mutant, most of them being proteins associated with the cytoplasmic membrane. Among suppressed proteins we detected the SbtA bicarbonate transporter (Slr1512, band 3), the UrtA urea transporter (Slr0447, band 4), the PilA1 member of pili family (Sll1694 band 5) and the RbcS small RUBISCO subunit (Slr0012, band 6) which seems to form a complex with SbtA. There was also one cytoplasmic protein upregulated and this was a phosphate binding transporter encoded by the sll0680 gene. In general, the data indicate that the Syn-CRY protein affects synthesis and accumulation of cytoplasmic membrane proteins including a fraction of the D1 protein destined for assembly of new PSII complexes which also is supposed to be synthesized in the vicinity of the cytoplasmic membrane [56].

3.5. PSII assembly in the absence of Syn-CRY The separation of thylakoid membrane proteins from radioactively labeled cells using two-dimensional clear-native/SDS electrophoresis (2D CN/SDS-PAGE) combined with autoradiography allowed us to assess in more detail the effect of the Syn-CRY

3.6. UV-B and HL induction of psbA genes in the presence or absence of Syn-CRY Under light stress conditions the transcription rate of the psbA genes that encode the D1 protein become significantly elevated

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Δ Sll1629

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2D stained gels

1D fluor

Fig. 4. 2D analysis of radioactively labeled thylakoid membrane protein complexes of WT and DSll1629 strains. Thylakoid membranes isolated from radioactively labeled cells grown autotrophically were analyzed by 2D CN/SDS-PAGE. The 1st dimension native gel was photographed (1D color) or scanned for Chl fluorescence (1D fluor) and after SDS-PAGE in the 2nd dimension the gels were stained by Coomassie (2D stained gel), dried and exposed to Phosphorimager plate (Autoradiograms). Designation of complexes: RCCS(1), PSII-PSI supercomplex; RCC(2) and RCC(1), dimeric and monomeric PSII core complexes, respectively; PSI(3) and PSI(1), trimeric and monomeric PSI; RC47, PSII core complex lacking CP43;U.P., unassembled proteins. Horizontal arrow designates incompletely processed form of D1 (iD1) in RCII assembly complexes of WT. The proteins differentially synthesized and accumulated in the mutant were designated with arrows and numbered: 1 – Slr0906 (CP47); 2 – Sll0680 (PstS); 3 – Slr1512 (SbtA); 4 – Slr0447 (UrtA); 5 – Sll1694 (PilA1); 6 – Slr0012 (RbcS). Vertical arrows associated with the numbers indicate either increased (arrow upwards) or decreased (arrow downwards) level of the protein in the mutant. The band of unassembled CP43 is circled. Each loaded sample contained 5 lg of Chl.

both under UV-B [57,58] and HL [58,59]. Transcription of psbA genes is required for the restoration of the UV-B or HL induced suppression of D1 protein level via de novo protein synthesis. In Synechocystis D1 protein is encoded by three psbA genes, among which psbA3 is specifically induced by UV-B [57] and also enhanced by high intensity visible light [60]. We made use of this marker to determine whether Syn-CRY exerts its beneficial effect to maintain PSII activity pre- or post-transcriptionally. We have found that during UV-B treatment psbA3 transcript levels in the WT and Dsll1629 cells were not significantly different from each other after 120 min (F = 0.247, at P = 0.05), or 270 min of UV-B treatment (F = 0.925, at P = 0.05) (Fig. 5). The same trend continued in the recovery phase where in the absence of UV-B the transcript levels dropped close to their initial values both in WT and mutant cells by the end of the 90 minute recovery period (F = 0.996, at P = 0.05). When cells were exposed to additional 60 min of UV-B radiation following the end of the recovery period both cell lines responded with a robust psbA3 induction (Fig. 5). However, significant differences were not detectable between the WT and the mutant (F = 0.994, at P = 0.05). This result shows that the inability of Dsll1629 cells to produce Syn-CRY does not affect their ability to maintain an adequate psbA3 mRNA pool, which is required for efficient D1 synthesis. 4. Discussion Since the discovery of the Cry-DASH family of proteins their physiological function has remained unresolved. Although these proteins have the potential to repair single stranded DNA [6,12– 14] they seem to be unable to function as conventional photolyases,

psbA3 relative transcript levels

RC C PS S(1 RCI(3) ) PS C(2 I(1 ) )

1) . C( C47 U.P C R R

RC C PS S( I 1 RC (3) ) PS C(2 I(1 ) )

WT CN - PAGE S D S P A G E

9

LL

UV-B

90

60

WT Δsll1629

8 7 6 5 4 3 2 1 0

0

120

270

Time (min) Fig. 5. The relative levels of psbA3 transcripts during UV-B stress and recovery, in Synechocystis cells. In three-phase experiments wild type and Dsll1629 mutant cells were first exposed to 8 lmol photons m 2 s 1 UV-B radiation together with 50 lmol photons m 2 s 1 of low intensity background white light (LL) for 270 min. In a second phase both cell lines were illuminated with 50 lmol photons m 2 s 1 of LL for 90 min as a recovery treatment from the UV-B stress. In the final phase 8 lmol photons m 2 s 1 of UV-B was administered to the cells to check for their repeated inducibility. The columns represent the induction levels of psbA3, after normalization to the transcript amount of the rnpB (slr1311) housekeeping gene of Synechocystis, coding for the RNase P subunit B. The data represent means obtained from 3 independent experiments with the indicated standard errors.

which repair UV-induced CPDs in double-stranded DNA. On the other hand, several lines of evidence show that members of the Cry-DASH family, including Syn-CRY from cyanobacteria function as photoreceptors [10,22,61,62]. Our recent study has revealed that photolyase-mediated repair of DNA is required for efficient PSII repair cycle in the cyanobacterium Synechocystis 6803, which proceeds via de novo synthesis of UV-damaged D1 subunit of the PSII reaction centre [38]. As a continuation of these investigations here we studied the potential role of the Syn-CRY photolyase homolog of Synechocystis 6803 (sll1629) in the repair of UV-B damaged PSII. 4.1. Effect of Syn-CRY on DNA repair DNA is highly sensitive to UV-B and upon irradiation specific, polymerase blocking lesions form in the molecule. Most of these lesions are cyclobutane pyrimidine dimers [63], which can be effectively reversed to native functional nucleotides by the photolyase enzyme (see Ref. [18]). In our previous study we have shown that an overwhelming majority of the UV-B induced lesions are efficiently repaired by PhrA in Synechocystis 6803 cells [38]. In agreement with this finding, which does not leave much room for an additional specific DNA repair activity, in the current study we could not detect unrepaired UV-B induced DNA damages in the DphrB cells. Our results corroborate previous studies, showing that although Syn-CRY does bind to DNA, this effect, unlike photolyase activity, is sequence independent [10] and most likely does not result in photoreactivation of CPDs [11]. In the DphrB cells we have found no detectable amount either of another frequently occurring DNA lesion, the 7,8-dihydro-8-oxoguanine (8-oxoG), which is not specific to UV-B stress, and related to oxidative stress. Thus, we conclude that Syn-CRY is not involved in the repair of either CPDs or the 8-oxoGs in Synechocystis 6803. 4.2. Effect of Syn-CRY on UV-B and HL induced loss of PSII activity An important target of UV-B radiation in the photosynthetic apparatus is the PSII complex in which electron transport is

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impaired and D1 and D2 reaction center subunits degraded (see [33,35]). In intact cells the extent of UV-B induced damage of PSII is determined by the balance between damaging processes and those restoring the structural and functional integrity of PSII complexes. Since UV-B induced loss of PSII activity proceeds with the same rate in the WT and Syn-CRY-less cells with the blocked de novo protein synthesis (Fig. 2) the presence or absence of the Syn-CRY protein does not influence the damaging effect of UV-B in the PSII complex. In contrast, in the absence of lincomycin which allows PSII repair, the larger extent of UV-induced loss of PSII activity in the Dsll1629 cells relative to the WT demonstrates that restoration of PSII function is retarded in the absence of Syn-CRY. The increased UV-B sensitivity of cells in the absence of Syn-CRY under conditions when protein synthesis is functional is also shown by the enhanced loss of D1 protein amount relative to the WT cells (Fig. 3). PSII damage and subsequent repair is induced not only by UV-B, but also by visible light. Interestingly the difference in PSII activity loss between the Syn-CRY deletion and WT cells can also be observed under elevated visible light (Fig. 2B). This finding points to the involvement of Syn-CRY in processes which are not specifically related to UV-B light, but affect the PSII recovery in a more general way.

4.3. Effect of Syn-CRY on PSII repair The data presented in Figs. 2 and 3 demonstrate for the first time that the lack of the Syn-CRY protein impairs the repair cycle of PSII, which is required to restore PSII activity following visibleor UV-light damage. This finding points to a so far unexpected role of Syn-CRY in photosynthetic processes of cyanobacteria. The PSII repair cycle plays a highly important role in keeping the PSII centers continuously operational, and any environmental effect which decreases its efficiency adversely affects photosynthetic performance [64,65]. The first main step of the PSII repair cycle is the proteolytic degradation of the damaged D1 (and D2) proteins (Scheme 1), in which the so called FtsH proteases play an important role in coping with photodamage induced by both UV-B [35] and visible light [66,67]. It is unlikely that the lack of Syn-CRY would affect directly or indirectly the degradation of D1 since D1 protein loss could be detected in the Syn-CRY lacking Dsll1629 cells (Fig. 3). The next important step of the repair cycle is the induction of psbA gene transcription in order to produce a sufficient level of psbA mRNA for D1 protein synthesis. Since induction of psbA3 transcription is also unaffected in the Dsll1629 mutant (Fig. 4) the absence of Syn-CRY does not seem to affect the gene transcription step of the repair process either. Following the synthesis of new D1 subunits, i.e. translation of psbA mRNA, the next important step of PSII repair is the assembly of PSII complexes, which was studied by 2D separation of the PSII subunits. The main effect of the lack of the Syn-CRY protein is the decrease in the amount of dimeric PSII complexes, as well as of the main PSII subunits (Scheme 1), which points to the influence of Syn-CRY either on translation of psbA (psbD) mRNA or the PSII assembly process. Importantly the amount of some PSII unrelated proteins was also decreased in the cells that lack Syn-CRY. From photoinhibitory point of view the most interesting among them are the SbtA, RbcS and the PilA1. The first protein is involved in bicarbonate transport and the second is the small subunit of RUBISCO, and both are important for CO2 fixation. Interestingly, these proteins seem to form a complex in Synechocystis (Fig. 3). The decreased level of both proteins indicates that the cells might suffer from CO2 fixation deficiency and it has been shown that inhibition of CO2 fixation leads to decreased rate of PSII repair due to the accumulation of reactive oxygen species which inhibit the synthesis of PSII proteins,

Scheme 1. Potential pathways for the influence of Syn-CRY mediated signaling events on the restoration of PSII activity. UV-B and high light damages the D1 (and D2) reaction center proteins of PSII. Restoration of PSII activity proceeds via degradation of the damaged subunits, followed by de novo protein synthesis and assembly of functional PSII RC complex. In the absence of the Syn-CRY photoreceptor the repair cycle is inhibited after the mRNA synthesis step. This inhibition can occur at the level of psbA translation due to accumulation of ROS, which is induced by decreased level of CO2 fixation in the absence of small subunit of Rubisco (RbcS) and of the SbtA component of the CO2 concentrating mechanism both of which appear to be under the control of Syn-CRY. Alternatively, the inhibition of PSII reactivation can occur at the assembly process via suppression of the synthesis of the PilA1 protein, which may be involved in delivery of Chl required for the functionalization of the PSII RC complex.

especially of D1 [68,69]. This effect is due to the inhibition of translation elongation by singlet oxygen and other ROS forms [64,70]. The involvement of Syn-CRY in regulating of the amount of the RbcS and StbA proteins is not yet clarified. However, low Ci inducible genes such as slr2013, which is part of slr2006-2013 cluster implicated in ion transport [71], are also regulated by Syn-CRY [10]. Thus it is feasible to assume that Syn-CRY is involved in Ci import and/or concentration processes of Synechocystis cells, whose impairment renders the cells sensitive to HL [72]. Another notable consequence of Syn-CRY deletion is the decreased amount of PilA1 (sll1694), which is a secreted protein that reaches the outer surface of the cell [73,74], and is involved in cell motility [75,76]. Interestingly, PilA1 has been implicated in binding or transferring Chl molecules to newly formed photosystems or antenna proteins [40]. Since Chl availability has been suggested to play a regulatory role at the assembly step during PSII repair [37,77,78], therefore, PilA1 could be involved in PSII repair as part of the Chl storage/delivery system. Although the pathway by which Syn-CRY could influence PilA1 biogenesis has not yet been clarified in detail one possible route could be through cAMP-mediated signal transduction. This idea is supported by the observation that disruption of the cya1 gene, encoding the adenylate cyclase enzyme of Synechocystis renders the cells immotile, which effect can be reversed by addition of extracellular cAMP [79]. It has also been shown that the lack of the a cAMP receptor protein SYCRP1 turns the cells non-motile [80], and also that the cAMP-SYCRP1 complex controls the pili biosynthesis [81]. Syn-CRY is apparently involved in the transcriptional repression of the ilvN (sll0065) gene [10], coding for acetolactate synthase, which has a putative binding site for the transcriptional regulator SYCRP1 [82]. Therefore, the absence of

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Syn-CRY could affect pilin biogenesis directly, or via the cAMP pathway leading to suppression of the PilA1 level, which in turn can inhibit PSII assembly by limiting Chl availability. 5. Concluding remarks The data presented here show for the first time that Syn-CRY protein has a specific physiological role in the regulation of PSII repair and its absence retards the restoration of UV-B and visible light induced damage of PSII in Synechocystis cells. Although SynCRY is a photolyase homolog, and the accumulation of unrepaired CPD lesions in the DNA has been shown to inhibit PSII repair [38], our data show that Syn-CRY does not repair CPDs and other DNA lesions (8-oxoGs) and its effect on the PSII repair cycle is related to the suppression of protein factors involved either in D1 (D2) protein synthesis or assembly of the PSII complex (Scheme 1). Possible candidates are proteins involved in CO2 transport and fixation like SbtA and RbcS. Alternatively, PilA1 (sll1694), a type IV pilin protein implicated in binding or transferring Chl molecules to newly formed photosystems or antenna proteins [40,83], could be involved in PSII repair as part of the Chl storage/delivery system. However, clarification of the details of this process requires further investigations. The main role of Syn-CRY is perception of blue light and initiation of blue light induced signaling pathways. The UV-A/blue region of the solar spectrum is an efficient inducer of PSII photodamage therefore this spectral range can also be utilized to trigger the repair processes via suitable photoreceptors. On the basis of the data presented here it appears that Syn-CRY has a specific physiological role in the regulation of PSII repair via regulating the level of CO2 transport and fixation proteins or the PilA1 protein. 6. Abbreviations

Chl CPD D1 and D2 DCMU PSII qPCR UV-A UV-B 6-4 PPs

chlorophyll cyclobutane pyrimidine dimer reaction center protein subunits of Photosystem II 3-(34-dichlorophenyl)-1,1-dimethylurea Photosystem II quantitative PCR ultraviolet-A (315–400 nm) spectral range ultraviolet-B (280–315 nm) spectral range thymine-thymine pyrimidine-pyrimidone (6-4) photoproducts

Acknowledgements This work was supported by the EU FP7 Marie Curie Initial Training Network HARVEST (Project No. 238017), by the TÁMOP4.2.2.A-11/1/KONV-2012-0047 project, by project Algatech (CZ.1.05/2.1.00/03.0110), RVO61388971 and P501/12/G055 of the Grant Agency of the Czech Republic. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2013. 12.006.

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