From the Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, P. O. Box 521, H-6701 Szeged, Hungary
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ABSTRACT |
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UV-B irradiation of intact Synechocystis sp. PCC 6803 cells results in the loss of photosystem II activity, which can be repaired via de novo synthesis of the D1 (and D2) reaction center subunits. In this study, we investigated the effect of UV-B irradiation on the transcription of the psbA2 and psbA3 genes encoding identical D1 proteins. We show that UV-B irradiation increases the level of psbA2 mRNA 2-3-fold and, more dramatically, it induces a 20-30-fold increase in the accumulation of the psbA3 mRNA even at levels of irradiation too low to produce losses of either photosystem II activity or D1 protein. The induction of psbA3 transcript accumulation is specific for UV-B light (290-330 nm). Low intensity UV-A emission (330-390 nm) and white light induce only a small, at most, 2-3-fold enhancement, whereas no effect of blue light was observed. Expression patterns of chimeric genes containing the promoter regions of the psbA2, psbA3 genes fused to the firefly luciferase (luc) reporter gene indicate that (i) transcription of psbA2/luc and psbA3/luc transgenes was elevated, similarly to that of the endogenous psbA genes, by UV-B irradiation, and that (ii) a short, 80-base pair psbA3 promoter fragment is sufficient to maintain UV-B-induced transcription of the luc reporter gene. Furthermore, our findings indicate that UV-B-induced expression of the psbA2 and psbA3 genes is a defense response against UV-B stress, which is regulated, at least, partially at the level of transcription and does not require active electron transport.
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INTRODUCTION |
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Recent reductions in the stratospheric ozone layer allowing more ultraviolet-B (UV-B, 280-320 nm) radiation to reach the Earth's surface and ecologically significant depths of the ocean (1) have initiated extensive research efforts to elucidate molecular mechanisms regulating responses of various organisms to UV irradiation (2). In photosynthetic organisms, a crucial part of the overall UV-B effect is related to damage in the photosynthetic apparatus leading to decreased oxygen evolution and CO2 fixation (3-6), reduction in dry weight, secondary sugars, starch, and total chlorophyll (7, 8). Within the photosynthetic apparatus, the most sensitive UV-B target is the light energy converting complex of photosystem II (PSII)1 (for reviews, see Refs. 9 and 10), although photosystem I (4, 11), ribulose-bisphosphate carboxylase/oxygenase (12, 13), and ATPase (14) can also be damaged. PSII is a large pigment-protein assembly embedded in the thylakoid membrane. Inhibition of PSII activity by UV-B radiation is accompanied by the degradation of the D1 and D2 protein subunits (15-18), which form the heart of the PSII reaction center by contributing ligands to cofactors that mediate primary photochemistry (for review, see Ref. 19).
Photoheterotrophic cyanobacterium species are very useful model organisms for studying the adverse effects of UV-B irradiation on the activity of PSII because (i) cyanobacteria were among the earliest oxygenic photosynthetic organisms that were exposed early in their evolution to UV-B fluxes much higher than the relatively low present day UV levels (20), and (ii) they have evolved efficient mechanisms to cope with the stress of UV exposure (21). Indeed, in a previous study, we have provided evidence that intact Synechocystis 6803 cells are able to repair UV-B-induced damage of PSII via de novo synthesis of the D1 and D2 proteins (22). Just recently, Campbell et al. (23) have reported that the cyanobacterium Synechococcus appears to resist UV-B-induced PSII damage by selectively exchanging the PSII reaction center D1:1 protein (encoded by psbAI) for the different D1:2 form (encoded by the psbAII and psbAIII genes). In Synechocystis 6803, the D1 protein is also encoded by three genes, psbA1, psbA2, and psbA3 (24). However, in contrast to Synechococcus, Synechocystis has only one form of the D1 polypeptide encoded by the psbA2 and psbA3 genes. The expression of both psbA2 and psbA3 is under light control (25, 26), with psbA2 accounting for the majority (>90%) of the psbA transcripts under normal growths conditions (26). The third psbA gene, psbA1, encodes a slightly different D1 protein (see Ref. 27), but there are no indications that it is expressed in wild-type cells (25, 26, 28).
In this study, we show that expression of the two active psbA genes of Synechocystis 6803, is induced, although differentially, in response to UV-B light. UV-B-induced elevation of the expression of the endogenous psbA genes or the chimeric psbA/luc genes is regulated, at least partially, at the level of transcription, and elevation of psbA transcript levels does not require de novo protein synthesis or active electron transport. These data suggest that transcriptionally regulated expression of the psbA2 and especially that of the psbA3 helps to maintain functional PSII under conditions of UV-B exposure and provide evidence for the role of duplicate psbA genes in cyanobacteria.
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EXPERIMENTAL PROCEDURES |
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Growth Conditions--
Synechocystis 6803 cells were
routinely grown at 40 µE m2 s
1 light
intensity in BG-11 medium at 30 °C, gently bubbled with air containing 5% CO2. Cells in the exponential growth phase
(A730 nm of 0.6-0.8) were harvested by centrifugation for
10 min at 4000 × g at room temperature. The
chlorophyll a content was determined by methanolic
extraction, and the cells were diluted to 30 µg of chlorophyll
a ml
1 by fresh BG-11 medium.
Light Source--
UV-B light was produced by a Vilbert-Lourmat
VL-215M lamp in combination with a 0.1-mm cellulose acetate filter
(Clarfoil, Courtaluds Chemicals, UK) in order to screen out any UV-C
contribution, below 290 nm. Under these conditions, our UV-B source
provided maximal emission at 312 nm with 60 µE m2
s
1 intensity at the surface of the cell suspension. For
some experiments, this intensity was decreased to 6 µE
m
2 s
1 by applying eight layers of cellulose
acetate filters. Due to the high optical density of the irradiated
samples, the average UV-B irradiance within the cell suspension was
attenuated by about a factor of 30, as calculated by taking into
account the absorption by the optically dense sample according to Ref.
29, corresponding to 2 and 0.2 µE m
2 s
1
average intensity in the above irradiation protocols, respectively. These intensities are in the physiologically relevant range, because in
full sunshine of about 2000 µE m
2 s
1 the
UV-B contribution is approximately 7 µE m
2
s
1.
Irradiation Conditions--
UV-B irradiation was performed in
open, flat glass containers in which 330 ml of cell suspension (30 µg
of cholorophyll/ml) of 15 mm depth was continuously stirred at room
temperature. Before UV-B treatment, the cells were incubated under
visible light of 40 µE m2 s
1 intensity
for 1 h. For monitoring the recovery process after UV-B exposure,
the irradiated cells were transferred to visible light of 40 µE
m
2 s
1 intensity and were kept at room
temperature under continuous stirring. Steady-state rates of oxygen
evolution were measured using a Hansatech DW2 O2 electrode
at a light intensity of 1000 µE m
2 s
1 in
the presence of 0.5 mM
2,5-dimethyl-p-benzoquinone as electron acceptor. D1 protein
analysis was performed as described previously by Sass et
al. (22).
mRNA Extraction, S1 Nuclease Protection Assay-- Total RNA was extracted as described by Nagy et al. (30). S1 nuclease protection analysis was performed according to Fejes et al. (31). Sequences of the synthetic single-stranded oligonucleotides employed in these assays were: (i) luc, 5'-ATAGAATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTACAGCTTTACATAT-3'; (ii) psbA2, 5'-CGCTGTTGGAGAGTCGTTGTCATTTGGTTATAATTCCTTATGTATTTGTCGATGTTCAGATTGGAACTGACTAAACTTAGTC-3'; (iii) psbA3, 5'-CGCTGTTGGAGAGTCGTTGTCATTTGGTTATAATTCCTTATGTATTTGTCAATGTTCAAAGGATTTGGCCTCAAGCTC-3'.
Recombinant DNA Work, and Construction of Chimeric Genes-- All DNA manipulations were performed according to Maniatis et al. (32). Genomic fragments containing sequences of the psbA2 and psbA3 genes upstream of the ATG start codon (530 and 580 base pairs, respectively) were amplified by PCR, subcloned into a pKS plasmid containing the luc reporter gene (a gift from Dr. G. Neuhaus-Url) as SalI-NcoI fragments, and sequenced. The resulting psbA2/luc and psbA3/luc chimeric genes were then inserted into the cyanobacterial expression vector plasmid pSB2A (a gift from Dr. Chauvat) and introduced into Synechocystis cells. Transgenic cells were selected according to Kreps et al. (33). 5'-Truncation of the psbA3 promoter and replacement of the psbA2, psbA3 leader regions in the chimeric genes were also performed by PCR using specific synthetic oligonucleotides. For detailed sequence information about the promoter regions used in these experiments, see Refs. 34 and 35.
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RESULTS |
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Differential Induction of the psbA Genes by UV-B Light--
Repair
of PSII via de novo synthesis of the D1 reaction center
protein following its damage by UV-B radiation is a multistep process
(22). Here, we studied in detail the first step of this process and
monitored changes in psbA transcript levels during UV-B
exposure and throughout the following recovery period. To ensure
gene-specific detection of the psbA2, psbA3 transcripts, synthetic oligonucleotides were employed in S1 nuclease
protection assays. First, we showed that UV-B radiation elevated the
expression level of the psbA genes. Fig.
1A (lanes 1-3)
shows that after 90 min of UV-B radiation the level of the accumulated
psbA mRNA increased about 5-7-fold. More importantly,
Fig. 1B (lanes 1-3) indicates that the level of
psbA3 mRNA was elevated about 20-25-fold. In contrast,
Fig. 1C (lanes 1-3) shows only a 2-3-fold
induction of the psbA2. When the UV-B-irradiated cells were
transferred back to white light of 40 µE m2
s
1, the amount of psbA3 transcripts decreased
again dramatically, whereas the amount of psbA2 transcripts
showed a more moderate, about 2-3-fold decrease (Fig. 1, B
and C, lanes 4-6).
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UV-B Specificity--
The signal transduction pathway that leads
to UV response of photosynthetic genes in cyanobacteria has not been
studied in detail, and no specific UV-B receptor has been reported yet.
However, data in the literature indicate that blue light of low fluence triggers a differential response in the psbA gene family in
Synechococcus sp. PCC 7942 (36). In order to exclude any
accidental effect of blue light, which might be emitted from our lamp,
as well as to probe the effect of other spectral ranges on the
psbA3 gene response, six different irradiation protocols
were compared. Exposure of cells, incubated at 10 µE m2
s
1 white light as control condition, to white light of
100 µE m
2 s
1 resulted in only a small
increase in psbA3 transcript level (Fig. 2A, lane 2).
Illumination of the cells with the same white light through a narrow
band blue filter with maximal transmission at 450 nm (Fig.
2B, dotted line) induced a response (lane
5) similar to that evoked by white light. Exposure of cells to
UV-B light, whose irradiance profile is shown in Fig. 2B
(solid line a) alone (lane 4) or in
combination with visible light (lane 3) led to the already
described (Fig. 1) immense induction of the psbA3 gene.
Combination of the UV-B light source with an UG-11 filter in order to
cut all blue and visible light in the 390-690-nm range, producing the
irradiance profile shown in Fig. 2B, line b
(dark shading), resulted in the same extent of
psbA3 induction (lane 6) as nonfiltered UV-B
light (lane 3). This excludes the possibility of an
accidental blue component of our lamp being responsible for the
induction of the psbA3 gene. The possible effect of the small but not negligible UV-A emission from the lamp in the 330-400-nm range was also checked. To this end, the UV-B component of the lamp was
blocked by a 330-nm cut-off filter, which produced the irradiance
profile shown by line c (light
shading) in Fig. 2B. Illumination with this light
protocol resulted in some enhancement of the psbA3
transcript level (lane 7), which was, however, much smaller
than that induced by UV-B (plus UV-A) light (lane 4). These
results demonstrate that the remarkable induction of psbA3 gene expression observed in response to illumination with our UV source
was specific for the UV-B component (290-330 nm) of the lamp, and
unrelated to the small UV-A emission or any accidental blue (visible)
illumination.
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Effect of Electron Transport Activity--
In order to check the
possible involvement of redox regulation in the UV-B-induced expression
of psbA3, two different experimental approaches were used.
In the first experiment, the intensity of UV-B light was decreased to
10% of that used in Fig. 1 and de novo protein synthesis
was blocked by the translation inhibitor lincomycin. Under these
conditions, the oxygen evolving activity was not decreased by up to 60 min of irradiation (data not shown). This indicates that the applied
UV-B light did not damage the function and protein structure of PSII
complexes, because in the absence of protein repair any damage should
have led to a decreased oxygen evolution rate. Surprisingly, even this
very low fluence of UV-B light was sufficient for a strong induction of
the psbA3 gene after a 10-min exposure, as shown in Fig.
3A. Because the very weak UV-B
light applied (average intensity within the cell suspension 0.2 µE
m2 s
1), was certainly below the limit of
driving PSII electron transport, this finding indicates that direct
redox control via reduction of the plastoquinone pool is unlikely to
play a role in UV-B induction of psbA3. This hypothesis was
further corroborated by another experiment, showing that inhibition of
electron transport through PSII by DCMU
(3-(3,4-dichlorophenyl)-1,1-dimethylurea) did not affect the induction
of psbA3 in response to UV-B light either. According to the
data in Fig. 3B, 60-min exposure of cells to UV-B light in
the absence (lane 2) and in the presence of DCMU (lane
4) induced psbA3 expression to similar extents.
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Reporter Gene Constructs-- To determine whether the UV-B-induced expression of the psbA3 and psbA2 genes is regulated at the level of transcription as well as to identify important regulatory regions, a series of chimeric genes were constructed and their expression in transgenic cyanobacteria cells analyzed. These chimeric genes contained various fragments of the 5'-upstream regions of the psbA2 and psbA3 genes fused to the luc gene encoding firefly luciferase. For these experiments, a low intensity UV-B light was used in order to ensure that gene expression is monitored under conditions when damage to PSII is minimal. The most important results obtained are summarized in Fig. 4. It was first established that the induction of the psbA2 and psbA3 genes followed the same pattern as observed under strong UV-B light (shown in Fig. 1), i.e. psbA3 mRNA accumulation was enhanced to 20-30-fold by UV-B irradiation and declined gradually to control levels during a 4.5-h recovery period. In contrast, psbA2 mRNA accumulation pattern showed only 2-3-fold changes either during induction or the recovery phase (Fig. 4, top left).
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DISCUSSION |
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In the present work, the effect of UV-B light on the expression of the Synechocystis psbA genes encoding the D1 protein was studied as an approach to gain information about the early steps of the protein repair process that was shown by Sass et al. (22) to be required for restoring the activity of the UV-B damaged PSII. It was found that Synechocystis 6803 cells respond to UV-B by the differential expression of the two active psbA genes, i.e. transcript levels of psbA3 were increased 20-30-fold in contrast to those of psbA2, which were increased only 2-3-fold. The immense induction of the psbA3 gene was highly specific for the UV-B spectral range (290-330 nm in our conditions), but this response was not influenced by the presence of background visible light (Fig. 2). Any major contribution to psbA3 induction by the low intensity UV-A component of our lamp or by an accidental blue illumination can be excluded. These data strongly suggest that up-regulation of the psbA3 gene can occur under natural conditions where UV-B light is always accompanied by visible illumination and provide the first demonstration for the existence of a specific UV-B-responsive gene in Synechocystis cells.
Besides the different extent of induction under UV-B exposure, the psbA2 and psbA3 transcript levels also showed different kinetic behavior during the course of UV-B and subsequent visible illumination. The 20-30-fold induction of psbA3 was more rapid, and after having reached a transient maximum exhibited a slight decline even under UV-B light. This decline was accelerated after transferring the cells to visible light, and the enhanced psbA3 transcripts decreased close to control levels after a 2-3-h recovery period (Fig. 1). In contrast, the 2-fold induction of psbA2 appeared to be slower, and the slightly higher levels induced by UV-B exposure were maintained for a longer period of time under the conditions of recovery in visible light. These observations support the idea that the elevation of psbA3 transcript level is a rapid response to UV-B light, which may be very important for cyanobacterial cells in coping with the stress situation caused by enhanced UV-B fluxes from solar radiation.
Our data on the chimeric reporter strains suggest that the UV-B-induced
differential accumulation of the psbA2 and psbA3
transcripts is likely regulated at the level of transcription for the
following reasons: (i) the expression pattern of the
psbA2/luc and psbA3/luc transgenes is identical
with those of the respective endogens and (ii) exchange of the
psbA2 and psbA3 leader sequences in the reporter
gene constructs does not affect the expression pattern (Fig. 4), which
places the site of the UV-B-responsive element(s) in the
psbA3 promoter. Furthermore, we show that truncation of the
psbA3 promoter region to 160 and
80 positions does not
eliminate the basic UV-B response. These latest observations indicate
that cis-regulatory element(s) responsible for UV-B-induced
transcription should reside within the first 80 bases of the
psbA3 promoter.
The psbA2 and psbA3 genes encode identical D1 polypeptides (the psbA1 gene is not expressed under conditions checked so far), and the physiological role of producing the same protein from two different genes is an intriguing open question. Based on our data, we postulate that the Synechocystis 6803 cyanobacterium regulates the repair of its-UV-damaged PSII reaction center by increasing the pool of psbA mRNA available for producing new D1 proteins by switching on the psbA3 gene, which we consider to be a UV-stress gene. This hypothesis is supported by the following observations; (i) under normal growth conditions, in visible light, the majority (at least 90%) of psbA transcripts arises from the psbA2 gene (25, 26) and most likely the majority of the D1 protein is also translated from these messages; (ii) mainly because of the highly induced transcription of the psbA3 genes by UV-B irradiation, the overall level of psbA mRNA is about 5-7-fold higher in UV-B-irradiated as compared with nonirradiated cells (Fig. 1A); and therefore it is likely (iii) that under these conditions the amount of D1 protein translated from the psbA3 message is also significantly increased. Considering, however, that even under the conditions of maximal UV-B induction the psbA3 and psbA2 transcript levels are more or less identical, it follows that a considerable amount of D1 protein mobilized for repair should also be translated from the psbA2 mRNA. Therefore, we suggest that the psbA3 gene serves as a fast-reacting backup copy that can be relied on under conditions of UV-B stress in order to comply with the demand for higher D1 protein production.
The utilization of duplicate psbA genes seems to be a common trait of various cyanobacteria. However, there are species, like Synechococcus 7942, that contain three active psbA genes, which produce two distinct forms of the D1 protein. The psbAI genes encodes the D1:1 protein, and the psbDII and psbDIII genes encode identical D1:2 proteins (37). Upon exposure to high intensity visible light, the constitutive D1:1 form is transiently replaced by the D1:2 form in the PSII reaction center (38). Moreover, parallel with our work, Campbell et al. (23) reported that UV-B irradiation of Synechoccocus 7942 cells also induces expression of the otherwise lowly expressed psbAII and psbAIII genes and leads to a transient exchange of D1:1 for D1:2 protein in the PSII reaction center. Furthermore, these authors found that constitutive over-expression of the psbAII and psbAIII genes renders the cells to be UV-B resistant, whereas deletion of these genes results in an increased UV-B sensitivity. Based on these observations, Campbell et al. (23) concluded that resistance to UV-B irradiation is mediated by the exchange of the D1:1 and D1:2 forms in Synechococcus cells. This explanation is clearly not applicable to Synechocystis cells, where the various psbA genes encode identical D1 proteins. To resolve this apparent contradiction, we propose the following hypothesis. In both Synechococcus and Synechocystis cells, resistance to UV-B irradiation is brought about mainly by the selectively induced transcription of one or two of the psbA genes. Induced transcription leads to an increased psbA mRNA pool that is used by the translation machinery to produce large amounts of new D1 proteins to replace the damaged D1 molecules in the PSII. Accordingly, we suggest that UV-B resistance in different cyanobacterial species is controlled mainly at the level of transcription, and the exchange of the various forms of D1 proteins merely represents the ratio of various psbA mRNAs available for translation.
Cyanobacteria were among the earliest oxygenic photosynthetic organisms, which most likely experienced much higher UV-B fluxes early in their evolution (2.5-0.6 × 109 years ago) than the relatively low present day UV levels. The development of a readily inducible spare gene copy for the D1 protein, located in the heart of the highly UV sensitive PSII complex, is most likely one of the strategies applied by cyanobacteria for coping with the stress of UV exposure. The fact that this gene arrangement has been maintained over 1-2 billion years indicates that UV stress has been a sustained condition during evolution. At present, the components of the signal transduction pathway or the UV-B receptor(s) mediating the UV-B-induced transcription of the psbA3, psbAII, psbAIII, or other genes in cyanobacteria are not known. Redox control of photosynthetic gene expression, including psbA, has recently been supported by various observations (41), pointing to the central role of electron flow through PSII that modifies the redox level of the plastoquinone pool, which is then transmitted by a not yet clarified mechanisms. Our results demonstrate the induction of psbA3 in the absence of PSII electron transport, observed at very low levels of UV-B light, or in the presence of the electron transport inhibitor DCMU (Fig. 3). This argues against a direct involvement of redox signaling in the transcriptional activation of the psbA3 gene under UV-B light. From these data, it is more likely that a specific UV-B-related signal transduction pathway is involved in the induction of psbA3.
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ACKNOWLEDGEMENTS |
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We thank Dr. Peter Nixon (Imperial College, London, UK) for the very kind gift of anti-D1 antibodies and Dr. Cornelia Spetea for help with D1 protein analysis.
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FOOTNOTES |
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* This work was supported by Hungarian National Scientific Research Grant OTKA-T 017049, Academy Research Grant AKP-9612-457 3, 3168, and Howard Hughes Medical Institute Grant 75195-542401.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be addressed.
Fax: 36-62-433-434; E-mail: imre{at}everx.szbk.u-szeged.hu.
1 The abbreviations used are: PSII, photosystem II; µE, microeinstein(s); PCR, polymerase chain reaction; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; nt, nucleotide(s).
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REFERENCES |
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