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UV-B irradiation of intactSynechocystis sp. PCC 6803 cells results in the loss of photosystem II activity, which can be repaired via de novosynthesis 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 ofpsbA2 mRNA 2–3-fold and, more dramatically, it induces a 20–30-fold increase in the accumulation of the psbA3mRNA even at levels of irradiation too low to produce losses of either photosystem II activity or D1 protein. The induction ofpsbA3 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 ofpsbA2/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 pairpsbA3 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 thepsbA2 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. UV-B irradiation of intactSynechocystis sp. PCC 6803 cells results in the loss of photosystem II activity, which can be repaired via de novosynthesis 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 ofpsbA2 mRNA 2–3-fold and, more dramatically, it induces a 20–30-fold increase in the accumulation of the psbA3mRNA even at levels of irradiation too low to produce losses of either photosystem II activity or D1 protein. The induction ofpsbA3 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 ofpsbA2/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 pairpsbA3 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 thepsbA2 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. 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 (1Smith R.C. Prézelin B.B. Baker K.S. Bidigare R.R. Boucher N.P. Coley T. Karentz D. MacIntyre S. Matlick H.A. Menzies D. Ondrusek M. Wan Z. Waters K.J. Science. 1995; 255: 952-959Crossref Scopus (793) Google Scholar) have initiated extensive research efforts to elucidate molecular mechanisms regulating responses of various organisms to UV irradiation (2Lao K. Glazer A.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5258-5263Crossref PubMed Scopus (114) Google Scholar). 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 (3Jones L.W. Kok B. Plant Physiol. 1966; 41: 1037-1043Crossref PubMed Google Scholar, 4Kulandaivelu G. Noorudeen A.M. Physiol. Plant. 1983; 58: 389-394Crossref Scopus (81) Google Scholar, 5Bornman J.F. Björn L.O. Akerlund H.-E. Photobiochem. Photobiophys. 1984; 8: 305-313Google Scholar, 6Vass I. Sass L. Spetea C. Bakou A. Ghanotakis D. Petrouleas V. Biochemistry. 1996; 35: 8964-8973Crossref PubMed Scopus (143) Google Scholar), reduction in dry weight, secondary sugars, starch, and total chlorophyll (7Basiouny F.M. Van T.K. Biggs R.H. Physiol. Plant. 1978; 42: 29-32Crossref Scopus (46) Google Scholar, 8Fiscus E.L. Booker F.L. Photosynth. Res. 1995; 43: 81-92Crossref PubMed Scopus (189) Google Scholar). Within the photosynthetic apparatus, the most sensitive UV-B target is the light energy converting complex of photosystem II (PSII) 1The abbreviations used are: PSII, photosystem II; μE, microeinstein(s); PCR, polymerase chain reaction; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; nt, nucleotide(s). (for reviews, see Refs. 9Bornman J.F. J. Photochem. Photobiol. 1989; 4: 145-158Crossref Scopus (274) Google Scholar and 10Vass I. Pessarakli M. Handbook of Photosynthesis. Marcel Dekker, Inc., New York1996: 931-950Google Scholar), although photosystem I (4Kulandaivelu G. Noorudeen A.M. Physiol. Plant. 1983; 58: 389-394Crossref Scopus (81) Google Scholar, 11Brandle J.R. Campbell W.F. Sisson W.B. Caldwell M.M. Plant Physiol. 1977; 60: 165-169Crossref PubMed Google Scholar), ribulose-bisphosphate carboxylase/oxygenase (12Strid A. Chow W.S. Anderson J.M. Biochim. Biophys. Acta. 1990; 1020: 260-268Crossref Scopus (273) Google Scholar, 13Nogués S. Baker N.R. Plant Cell Environ. 1995; 18: 781-787Crossref Scopus (98) Google Scholar), and ATPase (14Zhang J. Hu X. Henkow L. Jordan B.R. Strid A. Biochim. Biophys. Acta. 1994; 1185: 295-302Crossref Scopus (32) Google Scholar) 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 (15Trebst A. Depka B. Z. Naturforsch. 1990; 45c: 765-771Crossref Scopus (73) Google Scholar, 16Greenberg B.M. Gaba V. Canaani O. Malkin S. Mattoo A.K. Edelman M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6617-6620Crossref PubMed Scopus (266) Google Scholar, 17Melis A. Nemson J.A. Harrison M.A. Biochim. Biophys. Acta. 1992; 1100: 312-320Crossref Scopus (190) Google Scholar, 18Friso G. Spetea C. Giacometti G.M. Vass I. Barbato R. Biochim. Biophys. Acta. 1993; 1184: 78-84Crossref Scopus (95) Google Scholar), which form the heart of the PSII reaction center by contributing ligands to cofactors that mediate primary photochemistry (for review, see Ref. 19Andersson B. Styring S. Curr. Top. Bioenerg. 1991; 16: 1-81Crossref Google Scholar). 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 (20Kasting J.F. Science. 1993; 259: 920-926Crossref PubMed Scopus (1041) Google Scholar), and (ii) they have evolved efficient mechanisms to cope with the stress of UV exposure (21Castenholz R.W. Garcia-Pichel F. Whitton B.A. Potts M. Ecology of Cyanobacteria: Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht1998Google Scholar). 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 (22Sass L. Spetea C. Máté Z. Nagy F. Vass I. Photosynth. Res. 1997; 54: 55-62Crossref Scopus (106) Google Scholar). Just recently, Campbell et al. (23Campbell D. Erikson M.-J. Öquist G. Gustafsson P. Clarke A.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 364-369Crossref PubMed Scopus (155) Google Scholar) have reported that the cyanobacteriumSynechococcus 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 thepsbAII and psbAIII genes). InSynechocystis 6803, the D1 protein is also encoded by three genes, psbA1, psbA2, and psbA3 (24Jansson C. Debus R.J. Osiewacz H.D. Gurevitz M. McIntosh L. Plant Physiol. 1987; 85: 1021-1025Crossref PubMed Google Scholar). 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 (25Mohamed A. Jansson C. Plant Mol. Biol. 1989; 13: 693-700Crossref PubMed Scopus (253) Google Scholar, 26Mohamed A. Eriksson J. Osiewacz H.D. Jansson C. Mol. Gen. Genet. 1993; 238: 161-168Crossref PubMed Scopus (118) Google Scholar), with psbA2 accounting for the majority (>90%) of the psbA transcripts under normal growths conditions (26Mohamed A. Eriksson J. Osiewacz H.D. Jansson C. Mol. Gen. Genet. 1993; 238: 161-168Crossref PubMed Scopus (118) Google Scholar). The third psbA gene, psbA1, encodes a slightly different D1 protein (see Ref. 27Svensson B. Vass I. Styring S. Z. Naturforsch. 1991; 46C: 765-776Crossref Scopus (146) Google Scholar), but there are no indications that it is expressed in wild-type cells (25Mohamed A. Jansson C. Plant Mol. Biol. 1989; 13: 693-700Crossref PubMed Scopus (253) Google Scholar, 26Mohamed A. Eriksson J. Osiewacz H.D. Jansson C. Mol. Gen. Genet. 1993; 238: 161-168Crossref PubMed Scopus (118) Google Scholar, 28Salih G.F. Jansson C. Plant Cell. 1997; 9: 869-878Crossref PubMed Scopus (44) Google Scholar). In this study, we show that expression of the two activepsbA 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 ofpsbA transcript levels does not require de novoprotein 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. Synechocystis 6803 cells were routinely grown at 40 μE m−2 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 chlorophylla ml−1 by fresh BG-11 medium. 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 m−2s−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. 29Rontó G. Fekete A. Gáspár S. Módos K. J. Photochem. Photobiol. 1989; 3B: 497-507Crossref Scopus (14) Google Scholar, corresponding to 2 and 0.2 μE m−2 s−1average 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−2s−1. UV-B irradiation was in in which of cell suspension μg of of was at room temperature. UV-B the cells were under light of 40 μE m−2 s−1 intensity for For the UV-B the irradiated cells were to light of 40 μE m−2 s−1 intensity and were at room under of oxygen evolution were a at a light intensity of μE m−2 s−1 in the of as electron D1 protein was as by Sass et al. (22Sass L. Spetea C. Máté Z. Nagy F. Vass I. Photosynth. Res. 1997; 54: 55-62Crossref Scopus (106) Google Scholar). was as by Nagy et al. F. Plant Kluwer Academic The Google Scholar). was according to et al. A. I. M. S. Nagy F. Plant Mol. Biol. 1990; PubMed Scopus Google Scholar). of the in these (i) (ii) psbA2, were according to al. T. J. Scholar). containing of thepsbA2 and psbA3 genes of the and were by PCR, into a containing the luc reporter gene G. and The and psbA3/luc chimeric genes were into the expression and cells were according to et al. S. F. C. J. M. P. Mol. Gen. Genet. 1990; Scopus Google Scholar). of promoter and of the psbA2, psbA3 regions in the chimeric genes were also by specific For about the promoter regions used in these experiments, see Refs. R. J. P. McIntosh L. Res. 1989; PubMed Scopus Google Scholar and J. P. B. Res. 1990; 18: PubMed Scopus Google of PSII via de novo synthesis of the D1 reaction center protein damage by UV-B radiation is a (22Sass L. Spetea C. Máté Z. Nagy F. Vass I. Photosynth. Res. 1997; 54: 55-62Crossref Scopus (106) Google Scholar). we in the of this and in psbA transcript levels UV-B exposure and the of the psbA2, psbA3 were in we that UV-B radiation the expression level of the psbA genes. that min of UV-B radiation the level of the mRNA about that the level ofpsbA3 mRNA was about In only a 2–3-fold induction of the the cells were to white light of 40 μE the of psbA3 transcripts decreased dramatically, whereas the of psbA2 transcripts a more about 2–3-fold In order to the of the two with PSII activity, the in and were by a and with in oxygen activity and D1 protein that UV-B irradiation the of oxygen the of electron activity PSII, and the of the D1 protein in a min of UV-B irradiation, both the of oxygen evolution and the of the D1 protein decreased to about of the control Under the conditions, the ofpsbA3 transcripts was as with that in control the cells to white the PSII protein and the oxygen activity were a the of oxygen evolution and the of the D1 protein about of the control In the phase of the the ofpsbA3 transcripts to levels to in the control In the level ofpsbA2 transcripts only to a and at the slightly level to The that to UV response of photosynthetic genes in cyanobacteria has not in and no specific UV-B has reported However, data in the indicate that blue light of low a response in the psbA gene sp. PCC J. Biol. 1994; PubMed Google Scholar). In order to any effect of blue which be our as as to the effect of on gene different irradiation were of at 10 μE m−2s−1 white light as control to white light of μE m−2 s−1 in only a increase in psbA3 transcript level 2 of the cells with the white light a blue filter with maximal at nm 2 a response to that by white light. of cells to UV-B irradiance is in 2 or in combination with light to the induction of the psbA3 gene. of the UV-B light source with filter in order to blue and light in the range, the irradiance in 2 in the ofpsbA3 induction as UV-B light the of blue of our lamp for the induction of the psbA3 gene. The effect of the but not UV-A emission the lamp in the was also this the UV-B of the lamp was by a which produced the irradiance by in 2 B. with this light in some of the level which much than that by UV-B light These results that the induction of expression in response to with our UV source was specific for the UV-B (290–330 nm) of the and to the UV-A emission or any blue In order to the of in the UV-B-induced expression of two different were In the the intensity of UV-B light was decreased to of that used in and de novo protein synthesis was by the Under these conditions, the oxygen activity was not decreased by to 60 min of irradiation not that the UV-B light not damage the and protein of PSII because in the of protein repair any damage have to a decreased oxygen evolution even this very low of UV-B light was sufficient for a induction of the psbA3 gene a as in A. the very UV-B light intensity within the cell suspension 0.2 μE m−2 was below the of PSII electron this that control via reduction of the is to a role in UV-B induction of was by that of electron PSII by not the induction of psbA3 in response to UV-B light to the data in exposure of cells to UV-B light in the and in the of psbA3 expression to the UV-B-induced expression of the psbA3 and is regulated at the level of transcription as as to a of chimeric genes were and their expression in cyanobacteria cells These chimeric genes various of the regions of the psbA2 and psbA3 genes fused to the luc gene encoding firefly For these experiments, a low intensity UV-B light was used in order to that gene expression is under conditions damage to PSII is The most results are in was that the induction of the psbA2 and psbA3 genes the as under UV-B light in psbA3 mRNA accumulation was to 20–30-fold by UV-B irradiation and to control levels a In accumulation only 2–3-fold either induction or the phase The two chimeric reporter containing the and psbA3 regions the fused to the luc gene and the expression patterns as for the genes, luc mRNA levels UV-B exposure and under white light for the psbA3/luc and only for the of promoter to and the transcription no effect on the UV-B-induced accumulation of the luc the to more Furthermore, of the psbA3 with that ofpsbA2 the psbA3/luc promoter is to the not either the UV-B-induced accumulation or the of luc mRNA levels the the of the ofpsbA2 with that of psbA3 in the expression of These data indicate that (i) UV-B-induced expression of psbA3 is regulated at the transcription (ii) the majority of or this defense response against stress be within a 80-base promoter and the of the psbA transcripts not have In the present the effect of UV-B light on the expression of the Synechocystis psbA genes encoding the D1 protein was as to about the early of the protein repair that was by Sass et al. (22Sass L. Spetea C. Máté Z. Nagy F. Vass I. Photosynth. Res. 1997; 54: 55-62Crossref Scopus (106) Google Scholar) to be for the activity of the UV-B was that Synechocystis 6803 cells to UV-B by the expression of the two active psbA transcript levels of psbA3 were 20–30-fold in contrast to of psbA2, which were only The induction of the was specific for the UV-B (290–330 nm in our but this response was not by the of light contribution induction by the low intensity UV-A of our lamp or by blue can be These data suggest that of the psbA3 gene can under conditions UV-B light is accompanied by and provide the for the of a specific gene the different of induction under UV-B thepsbA2 and psbA3 transcript levels also different the of UV-B and The 20–30-fold induction of psbA3 was more and a a even under UV-B light. was the cells to and the transcripts decreased to control levels a In the induction ofpsbA2 to be and the slightly higher levels by UV-B exposure were for a of under the conditions of in light. These the that the elevation of psbA3 transcript level is a response to UV-B which be very for cells in with the stress by UV-B fluxes data on the chimeric reporter suggest that the UV-B-induced accumulation of the psbA2 and is regulated at the level of transcription for the (i) the expression of and psbA3/luc transgenes is identical with of the and (ii) of thepsbA2 and psbA3 in the reporter gene does not the expression which the of the in Furthermore, we show that of promoter to and does not the UV-B These indicate that for UV-B-induced transcription within the of The psbA2 and psbA3 genes identical D1 gene is not expressed under conditions and the role of the protein two different genes is on our we that the Synechocystis 6803 cyanobacterium the repair of PSII reaction center by the of psbA mRNA for D1 proteins by on the psbA3 gene, which we to be a gene. is by the (i) under normal growth conditions, in the majority least of the psbA2 gene (25Mohamed A. Jansson C. Plant Mol. Biol. 1989; 13: 693-700Crossref PubMed Scopus (253) Google Scholar, 26Mohamed A. Eriksson J. Osiewacz H.D. Jansson C. Mol. Gen. Genet. 1993; 238: 161-168Crossref PubMed Scopus (118) Google Scholar) and most the majority of the D1 protein is also these (ii) because of the transcription of the psbA3 genes by UV-B irradiation, the overall level ofpsbA mRNA is about higher in as with cells and it is that under these conditions the of D1 protein the psbA3 is also that even under the conditions of maximal UV-B induction the psbA3 transcript levels are more or it that a of D1 protein for repair also be the psbA2 we suggest that the psbA3 gene as a that can be on under conditions of UV-B stress in order to with the for higher D1 protein The of duplicate psbA genes to be a of various cyanobacteria. However, there are that three activepsbA genes, which produce two of the D1 protein. The genes encodes the D1:1 and and genes identical D1:2 proteins J. R. J. PubMed Scopus Google Scholar). exposure to high intensity the D1:1 form is by the D1:2 form in the PSII reaction center A.K. A. Gustafsson P. G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). with our al. (23Campbell D. Erikson M.-J. Öquist G. Gustafsson P. Clarke A.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 364-369Crossref PubMed Scopus (155) Google Scholar) reported that UV-B irradiation cells also induces expression of the expressed and and to a of D1:1 for D1:2 protein in the PSII reaction Furthermore, these that of the genes the cells to be UV-B whereas of these genes results in UV-B on these Campbell et al. (23Campbell D. Erikson M.-J. Öquist G. Gustafsson P. Clarke A.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 364-369Crossref PubMed Scopus (155) Google Scholar) that to UV-B irradiation is by the of the D1:1 and D1:2 in is not to the various psbA genes identical D1 proteins. this we the In both to UV-B irradiation is about by the selectively transcription of one or two of the psbA genes. transcription to psbA mRNA that is used by the to produce large of D1 proteins to the D1 in the we suggest that UV-B in different species is at the level of transcription, and the of the various of D1 proteins the of various for were among the earliest oxygenic photosynthetic organisms, which most much higher UV-B fluxes early in their evolution × than the relatively low present day UV The of a gene for the D1 in the heart of the UV sensitive PSII is most one of the by cyanobacteria for with the stress of UV The that this gene has that UV stress has a the of the or the UV-B the UV-B-induced transcription of the or genes in cyanobacteria are not control of photosynthetic gene has by various J.F. K. G. Curr. Biol. 1995; PubMed Scopus (81) Google Scholar), to the role of electron PSII that the level of the which is by a not results the induction of psbA3 in the of PSII electron at very low levels of UV-B or in the of the electron against a of in the of the psbA3 gene under UV-B light. these it is more that a specific is in the induction We UK) for the very of and Spetea for with D1 protein
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