Redox control of psbA expression in cyanobacteria Synechocystis strains

Journal

of

Photochemistry and Photobiology ELSEVIER

B:Biology

J. Photochem.Photobiol. B: Biol. 48 (1999) 104--113

Redox control of psbA expression in cyanobacteria Synechocystis strains Miguel Alfonso t, Irene Perewoska, Sabine Constant, Diana Kirilovsky * URA 1810 CNRS, 'Photordgulationet Dynamique des Membranes"V~g~tales'. Ecole Normale Sup~rieure, 46 rue d'Ulm, F-75230Paris, Cedex 05, France Received 9 August 1998; accepted l0 November 1998

Abstract The D l reaction-centre protein of the Photosystem II complex is very sensitive to light. It is continuously damaged, degraded and synthesized. The respective rates of these three processes are regulated by the light intensity. The means by which light regulates the expression of the psbA gene encoding the DI protein in cyanobacteria is still an open question. Our results demonstrate that photosynthetic electron transport has an important role in psbA expression in Synechocystis cells. Under steady illumination, addition of 3-( 3,4-dichlorophenyl )- 1,1 -dimethylurea (DCMU) or 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) induces a transient activation of psbA transcription. Transcription of other photosynthetic genes like psaE and cpcBA, respectively encoding the PSA-E subunit of Photosystem I and the [3 and subunits of phycocyanin, one of the components of the phycobilisome, decreases under the same experimental conditions. Prolonged incubation with DCMU (or DCMU + methyl-viologen) results in a progressive decrease ofpsbA transcription and an increased stability of the transcript. Our data point to a control mechanism that involves two different signals: accumulation of Q A specifically activates psbA transcription, whereas oxidation of the electron transfer chain downstream of photosystem II, most probably the plastoquinone pool and/or the cyt b~,f, decreases the expression of psbA and that of other photosynthetic genes like psaE and cpcBA. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Cyanobacteria;Gene expression; Redox control

1. Introduction Light, beyond being the driving force of photosynthesis, is also a major source of stress to photosynthetic organisms. It is well known that Photosystem II (PS II) is damaged by light. Under moderate light the rate of damage is balanced by the rate of repair and an optimal efficiency of photosynthesis is maintained. However, above a given light intensity, which is different for each organism and each growth condition, the rate of damage is no longer matched by the rate of repair and PS II activity, and hence photosynthesis, is decreased or completely abolished (for recent reviews on photoinhibition, see Refs. [ 1-4]. The light stress may lead to the death of photosynthetic organisms. In order to survive, plants and algae have evolved an intricate process for the repair of the damaged photosynthetic protein complexes. The D1 protein, which is one of the reaction-centre proteins of PS II, has a very important role in oxygenic photosynthetic organisms. Not only is it the binding site of essential cofactors for the *Corresponding author. Tel.: 33-1-44323540; Fax: 33-1-44323935: E-mail: [email protected] Present address: Dpto.de NutricionVegetal, E.E. 'Aula Dei', CSIC,Apdo 202. E-50080Zaragoza, Spain.

reduction of quinones and oxygen production, but it also serves as a 'fuse' during cell illumination or light stress. Under illumination, this protein is inactivated and then degraded. D1 is damaged at all light intensities and it has to be replaced [ 5-7 ]. At low and moderate light, D 1 is removed and replaced as fast as it is damaged. The turnover of DI increases with light intensity, but at high light intensity the damage is so fast that the repair can no longer keep up with it and inhibition of PS II activity is observed. When stressed cells are placed under growth-light conditions they recover their photosynthetic activity by replacing the damaged D1 protein [ 6,8,9]. The regulation of the expression of the psbA gene, encoding the DI protein, is essential for the survival of the photosynthetic organisms. The psbA gene is highly conserved in all higher plants, algae and cyanobacteria. It is usually present as a single-copy gene in the chloroplast genomes of plants and eukaryotic algae [ 10]. In mature chloroplasts, the regulation of psbA expression is mainly post-transcriptional [ 1 1]. Recent work, carried out in the green alga Chlamydomonas reinhardtii, demonstrated that nuclear encoded factors that interact with the 5' untranslated region ( 5 ' - U T R ) of psbA mRNA and other photosynthetic genes enhanced their translation

1[/11-1344/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved. PIISIOll-1344(99)OOO38-X

M. Alfonso et al. / J. Photochem. Photobiol. B: Biol. 48 (1999) 104-113

[ 12,13]. Light seems to regulate psbA translation by modulating the binding of these nuclear-encoded proteins to the mRNA depending on the ADP levels and the redox potential of the cell [12,14,15]. In cyanobacteria, light essentially modulates the transcription of the psbA gene, which is generally present as a family of genes [ 16]. Most of the studies have been carried out in Synechococcussp. PCC 7942 and to a lesser extent in Synechocystis sp. PCC 6803 and 6714 strains. These three strains have three psbA genes: psbAl, psbAH and psbAllI. In Synechococcus sp. PCC 7942, the divergent psbAI copy encodes form I of D 1 that is present at low light and the psbAII and psbAllI copies encode form II of the protein present at high light intensities [ 16-18]. D1 form II has a higher intrinsic resistance to photoinhibition and renders the reaction centres more photochemically efficient [18,19]. In contrast, in Synechocystis sp. PCC 6803 and 6714 thylakoid membranes, there exists a unique form of D 1 encoded by thepsbAHandpsbAlll genesthat are almost identical. Nevertheless, these two copies are differentially expressed; about 95% of psbA transcripts originate from psbAH, while only 5% originate frompsbAlll in cells grown at low-light conditions [20,21]. Although psbAH mRNA is also the major transcript detected under high-light conditions, the level of both transcripts, psbAH and psbAIll, increases after cell transfer from low to high light intensities [20,22]. The psbAl copy is never expressed [ 22]. Light can be sensed by specific photoreceptors such as the blue-light receptor or phytochrome and/or via redox sensors of the photosynthetic electron transport chain (for reviews, see Refs. [23-25] ). Our understanding of the mechanism by which white light regulates psbA expression in cyanobacteria is still poor. Different models have been proposed. Tsinoremas et al. [ 26] reported that, in the cyanobacterium Synechococcus PCC 7942, the expression of the different copies of the psbA gene is regulated via a blue-light photoreceptor. Another group has reported that in the same strain, the expression of the differentpsbA copies is modulated without changing light quality or intensity by lowering the culture growth temperature [27]. They interpreted the results as a redox control ofpsbA expression. In Synechocystissp. PCC 6803, it has been proposed thatpsbA mRNA stability, but not psbA transcription, is influenced by photosynthetic activity [28]. Finally, other authors have suggested that DI degradation products may regulate psbA gene expression at both transcriptional and translational levels in Synechoo,stiscells [29]. Nothing is known about light regulation of psbA expression in other cyanobacterial strains. We have used Synechocystis PCC 6714 [9] as a model organism to study the mechanism of PS II photoinhibition in cyanobacteria. Recently, we studied in Synechocystis PCC 6714 cells the behaviour of the transcription and translation machineries during light stress to determine the nature of the threshold for the inactive irreversible state of photoinhibition [30]. We showed that light stress induces a large increase of thepsbA-mRNA level due to an increase ofpsbA transcription initiation. During the light stress, the high rate of psbA

105

transcription is maintained for a long time. However, when the light stress is too long, psbA transcription also decreases. Nevertheless, a high level ofthepsbA transcript is maintained thanks to an increased stability of the psbA-mRNA. The results suggested that damage in the transcription and translation machineries is responsible for the inability of the cells to recover after a prolonged light stress. The aim of the present work was to determine whether a specific electron carrier of the electron transport chain is involved in the light regulation ofpsbA expression. To reach our objective, we varied the rate of electron transport through the photosynthetic electron transport chain by the addition of different inhibitors and electron acceptors. We measured the levels of the psbA transcript and the stability ofpsbA-mRNA under such different conditions. We also studied the behaviour of thepsaE and cpcBA transcripts, respectively encoding the PSA-E subunit of Photosystem I (PS I) and the [3 and tx subunits of phycocyanin, one of the components of the phycobilisome. The interest of this analysis resides in the fact that these proteins have a low turnover that is not light dependent and are more stable than D1 under light-stress conditions [9,30].

2. Materials and methods

2.1. Strain culture conditions and light treatments Wild-type Synechocystis sp. PCC 6714 cells were grown in a mineral medium as described in Ref. [ 31 ] with twice the concentration of nitrate. Cells were grown in a rotary shaker ( 120 rpm) at 30°C under a 5% CO~-enriched atmosphere and continuous illumination from fluorescent white lamps giving an intensity of about 90 p~E m - 2 s J. Synechocystissp. PCC 6714 cells were harvested by centrifugation and resuspended in fresh sterile culture medium containing 50 mM HEPES (pH 6.8) at a final concentration of 15 Ixg Chl ml-~ (or 30 ixgChl ml - 1). They were preincubated under low light (90 p~E m 2 s - ~, 30°C) for 30 min and then maintained at the same light intensity or transferred to high light. Under highlight conditions cells were incubated at 30°C in a thermostatted glass cuvette (3 cm diameter) under gentle stirring and illuminated with four 150 W Atralux spots, each one giving a light intensity of about 1000 ~xE m 2 s-~. When indicated, 3-(3,4-dichlorophenyl)- 1,l-dimethylurea (DCMU, 0.3 or 20 I~M), 2,5-dibromo-3-methy]-6-isopropyl-benzoquinone (DBMIB, 5 or 15 txM) and methyl-viologen (MV, 300 txM or 2 mM) were added. Rifampicin was used as an inhibitor of transcription to determine the stability of transcripts. It was added in excess at a final concentration of 300 ~g ml ] in order to avoid problems of antibiotic degradation. Rifampicin is orange, therefore it significantly absorbs in the blue-green region (between 400 and 500 nm). When taking an absorption spectrum of cells plus rifampicin at the concentration ratio used in the experiments, we observed that the absorbance in the 400-500 nm region was twice that of cells in the

106

M. AlJbnso et al, / J. Photochem. Photobiol, B: Biol. 48 (1999) 104-113

absence of rifampicin. Rifampicin does not absorb in the orange-red region of the spectrum. In consequence, the phycobiliproteins are similarly excited in the presence and absence of rifampicin while the chlorophyll is less excited in the presence of rifampicin. The difference in light intensities used in this study is so big that a variation in the mRNA stability induced by high light should be seen despite the rifampicin effect. Samples were collected at different times and cells were immediately pelleted and frozen in liquid nitrogen. All samples were stored at - 80°C until used.

2.2. RNA methods Total RNA was isolated using hot phenol and LiC1 as precipitating agent [22]. Once isolated, the total RNA concentration was spectrophotometrically determined and samples were stored at - 80°C. For Northern blot experiments RNA samples were denatured for 3 min at 70°C and separated by electrophoresis on a 1.2% agarose gel containing tbrmaldehyde as denaturing agent, l0 jxg of total RNA were loaded on each lane. The gel was transferred onto a charged nylon membrane ( H y b o n d - N ~, Amersham, UK) by capillary blotting and fixed to the membrane by 5 rain UV exposure and 2 h at 80°C. Blots were hybridized with different radioactive probes at 42°C for psbA and psaE and at 40°C for cpcBA and 16S. rRNA Northern blots were exposed to X-ray film (Kodak) to obtain autoradiograms.

littoralis (gift from Dr C. Passaquet). The probes were radiolabelled by the random priming method using the multiprime DNA labelling system (Amersham, UK). 2.4. Fluorescence and oxygen measurements Chlorophyll fluorescence parameters and oxygen evolution were measured simultaneously at 30°C, with a modulated fluorometer (PAM 101 chlorophyll fluorometer; Walz, Effelrich, Germany) adapted to a DWI Hansatech oxygen electrode [34]. The actinic light used for measurements was similar in intensity (90 wE m - 2 s - ~) to that used in the RNA experiments. The cells were not CO2-1imited during measurements. The yield of chlorophyll fluorescence was continuously monitored using a modulated low-intensity non-actinic light. Fo was measured in dark-adapted cells in the absence of any actinic light. Saturating multiple-turnover white-light pulses (3200 IxE m - 2 s - 1) were applied to assess the maximal fluorescence yield (Fm and Fro' ). The pulses were generated by an electronic shutter (Uniblitz, Vincent, USA) put in front of a KL- 1500 quartz-iodine lamp (Schott, Mainz, Germany) that was continously on. The shutter was controlled by the accessory module PAM-103. Photochemical quenching (qp) was calculated according to Ref. [35]. Gross oxygen evolution was calculated as net evolution during illumination plus dark consumption.

2.3. Hybridization probes

3. Results

2.3.1. psbA Probe A 0.7 kb Kpnl-KpnI fragment containing the psbAll gene region of Synechocystis sp. PCC 6714, encoding the 3' half

3.1. Behaviour of psbA, psaE and cpcBA transcripts after cell transfer from low to high light intensities

of the gene which contains the sequence of the QB niche, was used as a homologous probe [ 32 ]. This probe recognized the two expressed psbA copies, psbAH and psbAlll. Due to the similarity between the two copies (including the upstream unstranslated region), it was impossible to construct specific probes for each copy.

We first determined the levels ofthepsbA, psaE and cpcBA mRNAs after cell transfer from a low light intensity (90 IxE m - 2 s ~) to a high light intensity (4000 t~E m - 2 s - ~). Total RNA extracted from cells incubated for 0, 10, 30 and 60 min under high-light conditions was subjected to Northern blot analysis. The level of the psbA mRNA dramatically increased in the first 10 rain of high-light incubation (Fig. l ). This high level of psbA mRNA was maintained throughout the incubation under high-light conditions. In contrast, the psaE and cpcBA mRNA levels decreased (Fig. 1). After 60 rnin of high-light incubation, the psaE mRNA band was no longer detectable even after a long exposure of the autoradiogram. The cpcBA mRNA was not detectable after 30 min with the heterologous probe we used. The variations in the transcript levels might result from changes in transcription activity or mRNA stability. The stabilities of the psbA and psaE mRNAs under low- and highlight conditions were determined by adding rifampicin, an inhibitor of transcription initiation. Under high-light conditions, the rifampicin was added after 10 rain of cell incubation under stress conditions. The stabilities of both transcripts were similar under low- and high-light conditions: 18 min forpsbA and 15 min for cpcBA (Fig, 2). These results sug-

2.3.2. psaE Probe A 0.35 kb Aval-Eco24I fragment, derived from the plasmid pBSPsaE (gift from Dr B. Lagoutte, F. Rousseau, Ph.D. Thesis) and containing the whole psaE sequence from Synechocystis sp. PCC 6803, was used.

2.3.3. cpcBA Probe A DNA fragment containing the coding sequences of the [3 and a subunits of phycocyanin 2 from the cyanobacterium Calothrix sp. PCC 7601 (gift from V. Capuano and Dr N. Tandeau de Marsac) [33] was used.

2.3.4. rRNA Probes To verify the equal loading of the gels, the membranes were always probed with a 1.8 kb Pstl-EcoRI fragment containing the 16S rRNA gene of the brown algae Pylaiella

M. A ![onso et al. / J. Photochem. Photobiol. B: Biol. 48 (1999) 104-113

loo q,

High light incubation (rnin) 0

10

30

60

IO0 o

0.3kb

z or

80

EE

70,

o.

60-

2~

.=__ "5

cpcBA

sbA

90

t" ''°1 psaE

107

1.6 kb

40 7J 30~

Fig. 1. Behaviour of the psbA, psaE and cpcBA transcripts under high-light illumination. Total RNA was isolated from Synechocvstis PCC 6714 cells (30 Ixg Chl ml i, 30oc) illuminated at 4000 ~E m 2 s 1for 0 (just before the shift to high-light intensity), 10, 30 and 60 min. 10 Fg of total RNA were loaded on each lane and separated on tbrn~aldehyde gels, transferred onto nylon membrane and then probed with thepsbA,psaE and cpcBA probes (described in Section 2.3).

gested that the changes in the steady-state levels of the different transcripts were due to variations in transcription activity: psbA transcription increased and psaE (and most probably also cpcBA) transcription decreased.

20

~

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A shift from low light to high light increases PSII activity and induces reduction of the electron transport chain and also QA- accumulation. To determine whether the redox state of a specific element of the electron chain was involved in the accumulation of the psbA mRNA upon cell transfer to high light intensities, different chemicals that artificially change the redox state of the electron carriers were used. DCMU increases the concentration of QA- under a given light regime by inhibiting the electron transfer from QA to QB. At the same time, DCMU inhibits the reduction of the PQ pool by PS II. DBMIB prevents plastoquinone reoxidation by the cyt bJinhibiting linear and cyclic photosynthesis. Depending on its concentration and on the light intensity, DBMIB can also induce photochemical accumulation of QA • In the presence of methyl-viologen (MV), a PS I electron acceptor, the PQ pool is reduced by PS II but the electron acceptors from PS I (like ferredoxin, FNR, or thioredoxin) are mostly oxidized. The presence of DCMU + MV inhibits linear and cyclic electron transport, provoking a large oxidation of the electron transport chain after the DCMU block. The kinetics of fluorescence induction indicated that DBMIB at the concentrations used in the RNA experiments (5 and 15 ~M DBMIB) does not directly block the electron transfer from QA to QR (data not shown). We then determined whether addition of 5 and 15 IxM DBMIB induced QA accumulation by inhibiting plastoquinol oxidation. This accumulation can be followed by fluorescence measurements in a PAM fluorometer. At room temperature, the yield of

~

0

LLI

3.2. Effect of photosynthetic electron transport inhibitors on psbA, psaE and cpcBA mRNA levels

50

50

*

-

"6 40

30T 0

5

10

15

20

25

30

35

Time after rifampicin addition (min) Fig. 2. Rate of disappearance of the psbA and psaE transcripts under lowlight (90 p~E m 2 s ], closed symbols) and high-light (4000/~E m 2 s i, open symbols) illumination. Rifampicin was added 10 rain alter the shift to high light. Data were obtained by densitometry of the autoradiograms. Estimated error: 8%.

fluorescence depends on the oxidoreduction state of QA. When QA is oxidized, a minimum level of fluorescence is observed (Fo) since the excitons are efficiently trapped by the open centres. When QA is fully reduced, the fluorescence level reaches a maximum (F,,) since the centres are closed and thus unable to trap excitons. Therefore, the fluorescence level is proportional to the Q A concentration [36]. Cells were illuminated with continuous white light (90 txE m - 2 s ~, 30°C) in the PAM fluorometer at a concentration of 15 ~g Chl ml -J. Oxygen evolution and fluorescence yield (F~') were followed during the illumination. F., F~/and Fro' were also determined. A rapid increase of the F~' level was produced immediately after addition of DBMIB, indicating the closure o f P S II centres and QA accumulation (Fig. 3). A new steady-state fluorescence level was attained after several

M. Alfonso et a l . / J. Photochem. Photobiol. B: Biol. 48 (1999) 104-113

108

(a)

Light Exposure (min)

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psaE Light Exposure (min)

Fo---

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400

500

Time of light incubation (sec) Fig. 3. Measurements of fluorescence yield by a PAM fluorometer under white-light illumination before and after addition of DBMIB. Synechocystis PCC 6714 cells (15 Ixg Chl ml J) were illuminated by an actinic light similar in intensity (90 IxE m -2 s ~) to that used in the RNA experiments. The weak measuring modulated light was maintained throughout the experiment. Saturating multiple-turnover white-light pulses (3200 ixE m- -" s 600 ms duration) were applied to assess the maximal fluorescence yield (F~,').

minutes. This level depended on the DBMIB concentration. The photochemical quenching parameter (qp = ( F r o ' - F , ' ) / F v ' ) , which reflects the extent of reduction of QA, was calculated. The qp value, which was 0.81 before the addition of DBMIB, decreased to 0.44 or to 0.33 after 10 min incubation in the presence of 5 or 15 IxM DBMIB. These results indicated that the proportion of open PS II centres decreased and the concentration of QA increased upon DBMIB addition. At the same time, oxygen evolution was inhibited by 50 and 75%, respectively.

3.2.1. Rapid effect of the photosynthetic inhibitors straight after addition We studied the effect of the different inhibitors on the levels of the psbA, psaE and cpcBA mRNAs and on their stability, straight after addition and after a prolongated cell incubation in their presence. The stabilities of the mRNAs were determined by adding rifampicin. Under a given light intensity (90 IxE m - 2 s - ' ) and in the absence of any change in light intensity, the level ofpsbA mRNA largely increased upon addition of DBMIB (15 IxM), DCMU (20 IxM) or DCMU + MV (300 IxM) (Fig. 4 (a) ). Similar results were obtained upon addition of 5 IxM of DBMIB or 0.3 IxM of DCMU (data not shown). The same treatment induced a

0

5

15

30

LL + DCMU LL + DBMIB

cpcBA Fig. 4. Underillumination,the rapideffect of photosyntheticelectrontransport inhibitors on psbA, psaE and cpcBA transcript levels. Synechoo'stis PCC 6714 cells ( 15 IxgChl ml - ~) were preilluminatedfor 30 min at 90 IxE m -2 s ~ of white light at 30°C. Then, the inhibitors DCMU (20 IxM), DCMU+ MV (300 IxM) or DBMIB ( 15 txM)) were added and total RNA was isolatedat 0, 5, 15 and 30 min after additionof inhibitors.10 i.tgof total RNA were loaded on each lane and separated on formaldehydegels, transferred ontonylonmembraneand thenprobed withthe psbA,psaEandcpcBA probes (describedin Section 2.3). (a) Northern blot analysisofpsbAtranscript levels upon addition of the different inhibitorsunder low-lightconditions, psbA mRNA levels in control cells without any addition are also shown. (b) Northernblot analysisof psaEand cpcBAtranscriptlevelsunder low-light (LL) conditionsupon additionof the differentinhibitors. rapid decrease in the levels of psaE and cpcBA transcripts (Fig. 4 ( b ) ) . We then determined whether the accumulation of the psbA transcript was due to an increase in transcript stability or transcription activity. Rifampicin and photosynthetic inhibitors were simultaneously added. The stability of the psbA mRNA was virtually unmodified during the first few minutes after the addition of the inhibitors. Therefore, the increase in the psbA mRNA levels seemed to be due to an activation ofpsbA transcription (Fig. 5).

3.2.2. Long-term effect of oxidation of the electron transport chain by DCMU + MV on psbA expression The increase in psbA transcription induced by DCMU or D C M U + M V was a transient effect. Fig. 6(a) shows the rates of degradation of the psbA mRNA when rifampicin was added 40 rain after the addition of the different inhibitors:

M. Alfonso et al. I J. Photochem. Photobiol. B: Biol. 48 (1999) 104-113

100

< z

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,~ 60

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10

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, -- ..... ~ 40

50

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Time after Rifampicin addition (min) Fig. 5. Disappearance of the psbA transcript under the same experimental conditions described in Fig. 4. Rifampicin and inhibitors were simultaneously added: low light without inhibitors ( • ) ; in the presence of DCMU (©), DBMIB (r~) or DCMU + M V ( • ) . Data were obtained by densitometry of the autoradiograms from two independent experiments.

( a ) Time of Light incubation in the presence of inhibitors (min)

109

D C M U alone, M V alone or D C M U + MV. The half-lives of the psbA m R N A s were 18 min in the presence of M V alone, 35 min in the presence of D C M U alone and 60 min in the presence of D C M U + MV. From 40 min to 2 h of cell incubation in the presence of inhibitors, the steady-state levels of thepsbA m R N A remained constant or decreased (in the presence of M V + D C M U ) (Fig. 6 ( b ) ). These results indicated that the oxidation of the electron transport chain induced a progressive stabilization of the psbA mRNA. They also suggested that a progressive diminution of psbA transcription was induced under these conditions. W e then tested whether the inhibition of linear and cyclic electron transfer by addition of MV + D C M U had an effect on the accumulation of the psbA m R N A upon cell transfer from low to high light intensities. Fig. 7 shows that this increase (due to the shift to high light) was inhibited when the cells had been preincubated for 40 min under low light with D C M U + M V before the shift to high light. The psbA m R N A level increased during the first 30 min of low-light

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Time after Rif addition (rain)

Time of Light incubation in the presence of inhibitors (min) 40 45 55 70100 130

+ MV

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+ DCMU + MV Fig. 6. Long-term effect of photosynthetic electron transport inhibitors on psbA expression. (a) Long-term effect of photosynthetic inhibitors on the psbA mRNA stability under low-light conditions (90 IxE m 2 s ~). Synechocystis PCC 6714 cells ( 15 txg Chl ml l) had been preincubated for 40 min, under 90 IxE m 2 s ~ of white-light illumination, in the presence of MV ( 2 mM) ( • ) , DCMU ( 20 I.tM) ( • ), or DCMU ( 20 IxM ) + MV ( 300 IxM ) ( • ) before the rifampicin was added and samples for RNA isolation were taken. (b) Long-term effect of the photosynthetic inhibitors on the steady-state levels of psbA mRNA. Total RNA was isolated from cells that were incubated under white-light illumination (90 txE m 2 s ' ), in the presence of MV (2raM), DCMU (20 I~M), or DCMU (20 ixM) + M V (300 I~M) from 40 to 130 min.

M. Alfonsoet al. / J. Photochem.Photobiol.B: Biol. 48 (1999)104-113

110 High Light

0

5

incubation(min)

15

30

tl

+ DCMU

+ MV

+ Rif + DCMU + M V + Rif

psbA Fig. 7. Effect of photosynthetic inhibitors on psbA mRNA accumulation upon cell shift to high light intensities. SynechocystisPCC 6714 cells (30 ~g Chl ml ~; 30°C) were transferred from low light (90 ~tE m ~ s ~) to high light (4000 ~E m 2s t) in the absence ofinhibitors or in the presence of DCMU (20 ~M) + MV (300 o,M). Cells had been preincubated for 40 rain in low light in the presence or absence of photosymhetic inhibitors before shifting the cells to high-light conditions. Rifampicin was added at the time of shifting the cells to high light. Blots were hybridized with the p~hAprobe. incubation (as already shown in Fig. 4) ; however, no further increase was observed upon shifting the cells to a higher light intensity. Again, inhibition of psbA accumulation by D C M U + M V was accompanied by an increase of psbA mRNA stability (Fig. 7).

4. Discussion In this report, we have studied the behaviour of the transcripts ofpsbA encoding D 1 and of two other photosynthetic genes, cpeBA, coding for the [3 and o~ subunits of phycocyanin, and psaE, encoding the subunit PSA-E of P S I . DI is particularly susceptible to damage by light and it has to be frequently replaced by a newly synthesized protein [ 1-4]. In contrast, the other proteins of the photosynthetic apparatus are less susceptible to light and they have a lower turnover. Two different sensory pathways by which light may control the expression of photosynthetic genes have been proposed. One involves wavelength-specific photoreceptors; the other one involves redox sensors of the photosynthetic electron transport chain. From the experiments presented in this paper, we cannot confirm or rule out a regulatory pathway involving a specific blue-light photoreceptor as proposed by others [26]. However, our results do not support a model where the expression ofpsbA would be controlled only by a blue photoreceptor. We demonstrated here that in Synechocystis PCC 6714, photosynthetic electron transport has an important role not only in the stability of psbA m R N A as previously suggested [28], but also in the regulation ofpsbA transcription.

The expression of the other two genes is also regulated by the redox state of the electron transport chain. The proposed involvement of redox sensors in the regulation ofpsbA gene expression is also valid for Synechoqvstis PCC 6803. The experiments described here were also carried out with Synechoqvstis PCC 6803 cells (data not shown). Similar results were obtained with these two strains, which are closely related. We only showed the results obtained in Syneehoqvstis PCC 6714, since this strain has been extensively used in our past studies on photoinhibition and herbicide resistance. When Synechoq~,stis cells are transferred from low light to high light, a sharp increase of the level of the psbA transcript is observed, while the steady-state levels of the psaE and cpcB-cpeA transcripts rapidly decrease. We have already demonstrated ( [30], see also Fig. 1 ) that the variations on the mRNA levels are due to changes in transcription activity. Under high light intensities, the degradation of the transcripts is rapid and similar to or even slightly faster than under low light illumination. Shifting the cells to high light intensities increases the psbA transcription activity and decreases psaE and cpcBA transcription activities. We cannot distinguish between psbAll and psbAlll mRNAs by Northern techniques. However, results obtained by primer extension analysis in Synechocystis PCC 6803 and 6714 ( [ 20], Alfonso and Kirilovsky, unpublished data) suggested that the two copies of the gene are similarly regulated by changes in white-light intensities. In Synechococcus PCC 7942, light also regulates the rate of transcription of psbA genes and the transcript stability. In this strain, each copy is differentially regulated: the shift to higher intensities induces an increase in the rate of transcription of the psbAH and psbAlll copies and a destabilization of the psbAl and psbAlll transcripts [ 17,18 ]. This differential regulation results in a replacement of form I by form II of D1 in the thylakoids, which leads to a decreased sensitivity to light stress [ 18,19]. In SynechocystisPCC 6803 and PCC 6714 only one form of D1 exists, since the divergent copy is not expressed and the homologous copies encode the same protein [ 20,21 ]. An increase in the rate of transcription and in that of translation is sufficient to replace the damaged D1 protein more frequently. From the studies carried out in Synechocystis PCC 6803 and PCC 6714 and Synechococcus PCC 7942, one can conclude that in cyanobacteria the increase of total psbA transcripts is a general response to a cell shift to higher light intensities. However, each strain presents its own characteristic regulations that cannot be generalized to the rest of the cyanobacterial strains,

4.1. Role of the redox state qf different electron carriers of the photosynthetic electron chain Our results clearly demonstrated that psbA, psaE and cpcBA gene expression can be modified by artificially changing the photosynthetic electron transport rate and the redox state of electron carriers, Under illumination, even in the absence of any change of light intensity, addition of DCMU or DBMIB induced accumulation ofpsbA mRNAs and dim-

M. A1J?msoet al. / J. Photochem.Photobiol.B: Biol.48 (1999)104-113 inution of the psaE and cpcBA mRNAs. Our results suggest that these changes on the mRNA steady-state levels were due to variations in transcription activity. The stability of the mRNAs was not modified in the first few minutes after addition of inhibitors. Our results suggest that transcriptional activation ofpsbA occurred whenever the QA- concentration was increased. Low-light to high-light shifts increase PSII activity and induce an increase of QA concentration. Room-temperature fluorescence measurements showed that addition of DCMU or DBMIB induced a rapid closure of PSII centres and QA accumulation. Moreover, QA- accumulation seems to be a specific signal for activation ofpsbA expression. Under illumination, addition of DCMU or DBMIB induced a decrease (instead of an increase) of the steady-state levels of the psaE and cpcBA transcripts. These results indicate that an increase in QA concentration is not sensed as an activation signal for psaE and cpcBA transcription. Since DCMU and DBMIB have opposite effects on the redox state of the plastoquinone pool, we conclude that psaE and cpcBA expressions are not directly correlated with the redox state of the plastoquinone pool. On the other hand, we do not rule out the possibility that the oxidation of one of the components of cyt b6fmight be the signal involved in the repression ofpsaE and cpcBA transcription. We have already shown that the levels of these transcripts also decreased under stress conditions ( [ 30] and Fig. 1 ). Under stress conditions, PS II is damaged, its activity decreases and cyt b6fbecomes more oxidized. Our hypothesis about the relationship between reduced QA and activation of psbA transcription predicts that in cyanobacteria, any modification affecting the rate of electron transport between QA- and QB will affect psbA gene expression. Indeed, a cell shift to higher light intensities induces a rise of QA concentration as well as an activation ofpsbA transcription. Results from other laboratories supporting this idea have also been described in the literature. Thus, it has been reported that growth ofSynechococcus PCC 7942 cells in the presence of sublethal concentrations of DCMU increases the steadystate levels of psbA mRNA [ 37 ]. Under growth conditions, the rate of psbA transcription is higher in mutants with a modified QA to Qs electron transport than in wild-type Synechocystis cells [29]. Furthermore, in Synechoccocus PCC 7942, cell-chilling treatments that induced an increase of the excitation pressure also induced an increase ofpsbAH and psbAIll mRNA levels [ 27]. The activation ofpsbA transcription occurring without any change in light intensity upon addition of DCMU was a transient effect. Prolonged cell incubation in the presence of DCMU and DCMU + MV caused a progressive inhibition of psbA transcription and an increase ofpsbA mRNA stability. In contrast, the presence of MV alone, even at a higher concentration, had no effect on psbA expression. The presence of DCMU + MV also inhibited the psbA mRNA accumulation induced by shifting cells from low-light to high-light conditions. These results could be explained by the antagonistic effect of DCMU on the redox state of QA and on that

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of the PQ pool and the cyt b6f'. DCMU increases the half-life of QA - and induces the oxidation of the PQ pool and the cyt b6fby inhibiting the electron transfer from QA tO QB. Our results also suggest that the redox state of the ferredoxin or thioredoxin might not have an important role in this regulation. The increase ofpsbA mRNA level induced by addition of DCMU or DBMIB, in the absence of any change in light intensity, may mask the long-term effect of the photosynthetic inhibitors. It has been reported that in Synechocystis PCC 6803, psbA mRNA levels increased when cells were transferred to higher light intensities even when electron transport was inhibited [ 28 ]. These authors compared the level ofpsbA mRNA before inhibitor addition with that after 3 h of highlight incubation in the presence of DCMU or DCMU + MV. Our results show that addition of DCMU, or DCMU + MV, induces an immediate increase ofpsbA transcript levels without any change in light intensity. Only after 30 min in the presence of these inhibitors is a progressive inhibition ofpsbA transcription and an increase in the stability of the transcript observed. In this work we preincubated the cells in the presence of the inhibitors for 40 rain and then determined the levels ofpsbA mRNA before and after the cell shift to high light in the presence of DCMU + MV. Under these conditions, the enhancement of the level of the psbA mRNA due to the shift to high light was inhibited. The double effect of DCMU on psbA expression (first stimulation and then down-regulation) suggests the involvement of two possible redox signals. Moreover, our results suggest that the activation signal is less sensed by cells where the electron transport chain is oxidized. When DCMU or DCMU + MV is added in the light, two different signals are given to the cell: PSII centres are closed so QA ~ is accumulated and, at the same time, electron carriers get oxidized. Because the closure ofcentres and QA accumulation occurs faster than the oxidation of the electron transport chain, since respiration continues to occur at least for half an hour (data not shown), a transient activation of transcription can be induced. However, once the repression signal appears, psbA transcription is decreased and psbA transcript stability increases. These results could be explained by the fixation of repressor proteins at specific promoter sites that block the RNA polymerase reaction even if activator factors are present. In prokaryotic organisms, overlapping effects of activation and repression of different genes allow flexibility of the regulatory apparatus to optimize adaptations to changing environment. The mechanisms governing these adjustments in cyanobacteria are largely unknown. Models for bacterial transcriptional control are founded on early studies of E. coli operons, where repressor and activator proteins were found to play a role in the transduction of environmental signals to the transcriptional apparatus. Several activator factors that specifically bind to psbA promoter regions in Synechococcus PCC 7942 have been characterized [38]. To our knowledge, no data are available for psbA activator factors in Synecho-

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cystis P C C 6714 or 6803. H o w e v e r , several s e q u e n c e s w i t h i n the p r o m o t e r r e g i o n o f the p s b A g e n e s d i s p l a y h o m o l o g i e s to r e p r e s s o r and activator fixation sites ( A l f o n s o and K i r i l o v sky, u n p u b l i s h e d d a t a ) .

Acknowledgements W e t h a n k Dr L a g o u t t e ( C E A , S a c l a y ) and D r N. T a n d e a u de M a r s a c (Institut Pasteur, P a r i s ) for p r o v i d i n g us w i t h the p B S P s a E p l a s m i d and the p l a s m i d p P M 6 2 , r e s p e c t i v e l y . W e also t h a n k G. P a r e s y s and J.P. R o u x for c o m p u t e r assistance. W e e x p r e s s our gratitude to A.-L. E t i e n n e for helpful discussions and critical r e a d i n g o f this m a n u s c r i p t .

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