Changes in photosynthetic electron transfer and state transitions in an herbicide-resistant D1 mutant from soybean cell cultures

14/02/2007 1 Changes in photosynthetic electron transfer and state transitions in an herbicideresistant D1 mutant from soybean cell cultures Mercedes Roncela*, Inmaculada Yruelab, Diana Kirilovskyc, Fernando Guerreroa, Miguel Alfonsob, Rafael Picorelb, José M. Ortegaa aInstituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Américo Vespucio 49, 41092-Sevilla, Spain. bEstación Experimental de Aula Dei, CSIC, Avda. Montañana 1005, 50059-Zaragoza, Spain. cLaboratoire de Bioénergétique Moléculaire et Photosynthèse, Institut de Biologie et Technologies-Saclay (iBiTec-S), CEA Saclay, 91191 Gif-sur-Yvette, France. *Author for correspondence: Mercedes Roncel Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Américo Vespucio 49, 41092-Sevilla, Spain Tel: 34 954 489525 Fax: 34 954 460065 E-mail: m[email protected] Keywords: cyclic and linear electron flow; Photosystem I, Photosystem II, thermoluminescence; state transitions; D1 mutant 14/02/2007 2 Abstract Anomalies in photosynthetic activity of the soybean cell line STR7, carrying a single mutation (S268P) in the chloroplastic gene psbA that codes for the D1 protein of the photosystem II, have been examined using different spectroscopic techniques. Thermoluminescence emission experiments have shown important differences between STR7 mutant and wild type cells. The afterglow band induced by both white light flashes and far-red continuous illumination was downshifted by about 4ºC and the Q band was upshifted by 5ºC. High temperature thermoluminescence measurements suggested a higher level of lipid peroxidation in mutant thylakoid membranes. In addition, the reduction rate of P700+ was significantly accelerated in STR7 suggesting that the mutation led to an activation of the photosystem I cyclic electron flow. Modulated fluorescence measurements performed at room temperature as well as fluorescence emission spectra at 77 K revealed that the STR7 mutant is defective in state transitions. Here, we discuss the hypothesis that activation of the cyclic electron flow in STR7 cells may be a mechanism to compensate the reduced activity of photosystem II caused by the mutation. We also propose that the impaired state transitions in the STR7 cells may be due to alterations in thylakoid membrane properties induced by a low content of unsaturated lipids. Abbreviations: AG, afterglow (luminescence bounce); B band, TL band due to S2/3QBrecombination; C band, TL band due to TyrD+QArecombination; FR, far-red light > 700 nm; HTL, high temperature thermoluminescence; PSI, photosystem I; PSII, photosystem II; Q band, TL band due to S2QArecombination; QA ,and QB, the primary and secondary quinone acceptors of the reaction centre of PSII; RC, reaction centre; S2 and S3, oxidized S2+ and S3+ states of the manganese oxygen-evolving complex of PSII, respectively; TL, thermoluminescence; tmax, temperature of the signal intensity maximum of a TL band 14/02/2007 3 1. Introduction The photosynthetic process operates according to two non-mutually exclusive modes in cyanobacteria, algae and plants: linear and cyclic electron flows. In the linear mode, electrons are transferred from water to NADP via the three major photosynthetic complexes, namely PSII, cytochrome b6f and PSI. Cyclic electron flow driven by PSI produces ATP, but not NADPH. Several alternative electron pathways driven by PSI have been reported to recycle electrons towards the intersystem carriers, mediated by either the NAD(P)H dehydrogenase (NDH complex) or the putative ferredoxin:plastoquinone reductase (FQR complex) [1-4]. Environmental stresses, such as photoinhibition, high temperatures, drought or high salinity, stimulate the activity of alternative PSI-driven electron transports. Thus, these pathways may provide additional flexibility to respond to different environmental stresses in plants [1]. Recently, it has been demonstrated that both FQR and NDH complexes are involved in the electron transfer reactions leading to a thermoluminescence (TL) band emission named “afterglow” (AG) [5]. AG emission has been related to cyclic electron transport, since the addition of an inhibitor of this flow, antimycin, suppressed this process [6]. Moreover, it has been observed that short heat-treatments, in the same temperature range which induces AG emission, trigger a PSI-driven electron flow from reducing components of unknown identity in the stroma towards the acceptor side of PSII [7]. This supports the idea that AG emission is due to the heat-induced reduction of QB by a stromal component and further recombination with S2/S3 states of the manganese cluster in PSII [8-10]. State transitions have been described as an adaptation mechanism that allows plants and algae to respond to changes in the spectral quality of light by varying the relative absorption cross-section of PSI and PSII [11,12]. This mechanism involves the reversible association of the major antenna complex (LHCII) with either PSII (in state 1) or PSI (in state 2) depending on the redox state of the intersystem electron carriers. LHCII phosphorylation by a membranebound protein kinase leads to the migration of a fraction of LHCII from PSII to PSI by lateral 14/02/2007 4 diffusion in the lipid phase. The LHCII kinase is activated by reduction of plastoquinone (PQ) pool (either by PSII activity or by other cellular metabolic processes) and inactivated by oxidation of PQH2 by PSI activity. Cytochrome b6f complex plays a key role in transduction of the redox signal from plastoquinol pool to kinase. Dephosphorylation of Pi-LHCII is catalysed by a phosphatase, which is supposed to be regulated by a 40 kDa luminal protein. Different authors have proposed that state transitions may be a mechanism of regulation of linear and cyclic electron transports. State 1 and state 2 correspond to two different modes of electron transfer in the green algae Chlamydomonas reinhardtii [11,12]. In state 1, PSII and PSI are functionally connected through the linear electron transfer chain, which generates NADPH and ATP. Upon transition from state 1 to state 2, redistribution of LHCII from PSII to PSI and migration of a fraction of the cytochrome b6f complex from the grana to the stroma thylakoid region induce cyclic electron flow around PSI at the expense of the linear electron flow and, thus, generating solely ATP [12]. STR7 is an atrazine-resistant mutant isolated from photosynthetic cell-suspension cultures of soybean, with a single mutation in the chloroplastic psbA gene coding for D1 protein of PSII core [13]. This mutation implies the substitution of serine 268 in D1 protein by proline (S268P) and it is different from the S264G or T mutations reported in other herbicide resistant biotypes. The STR7 strain showed some important differences as compared to the wild type (WT): slower growth, reduced oxygen-evolving activity, reduced electron transfer rate between the secondary acceptors QA and QB, presence of higher amounts of non-QB-reducing PSII centres and a larger antenna size [13]. Two other very interesting characteristics of STR7 mutant are its unusual tolerance to high temperatures and its increased sensitivity to light stress [14,15]. Both properties seem to be related to the alteration of lipid composition found in STR7. This mutant strain showed an unusually high content of saturated (and reduced levels of unsaturated) fatty acids in comparison with WT and, consequently, a more rigid thylakoid membrane matrix [14,16]. Changes in fatty acids unsaturation have also been reported for 14/02/2007 5 many other plant atrazine-resistant mutants, however, on the contrary, these mutants displayed higher levels of unsaturated lipids [17-20]. In this article, we report new data on the photosynthetic characteristics of the STR7 atrazine-resistant mutant. Significant alterations in PSII activity, state transitions and balance between linear and cyclic electron flows have been observed. 2. Materials and methods 2.1. Cell growth conditions Two different cell culture lines from soybean (Glycine max L. cv. Corsoy) were used in this study: SB-P line, kindly provided by Prof. J.M. Widholm (Department of Agronomy, University of Illinois at Urbana, USA), here denoted as the WT strain, and STR7 mutant, obtained from SB-P line by selection against s-triazine herbicide [13]. Both cell lines were grown in solid media on 1.5% (w/v) agar plates in KN1 medium under continuous low light (10 ± 5 µE m-2 s-1) at 24ºC and 5% CO2 atmosphere [21]. Cells grown in liquid culture were maintained in a climate chamber where they were grown at 25ºC under continuous light at 25 µE m-2 s-1 before the measurements. Just before the assays, both cell lines were suspended in 50 mM Mes-NaOH (pH 6.5) buffer. 2.2. Thermoluminescence measurements Thermoluminescence (TL) glow curves of soybean cell suspensions were measured using two similar home-built apparatuses designed by Dr. Ducruet (Saclay, France) for luminescence detection from 0ºC to 80ºC (standard TL) and from 10ºC to 160ºC (high temperature thermoluminescence, HTL). A detailed description of these systems can be obtained elsewhere [22,23]. Typically, samples were dark-incubated for 2 min at 20oC, then cooled to 1oC for 1 min and illuminated at the end of this period with different numbers of saturating single turn-over flashes (separated by 1 s) of white light through an optic fibre. 14/02/2007 6 Luminescence emission was recorded while warming samples from 0oC to 80oC at a heating rate of 0.5oC per s (TL) or from 10ºC to 160ºC at a heating rate of 0.1ºC per s (HTL). N2 gas was flushed on the sample during HTL experiments in order to desiccate samples and prevents any oxidation induced by high temperatures. The instruments were driven by a PC computer, with a specially developed acquisition program [24]. Data acquisition, signal analysis and graphical simulation were performed as previously described [23,24]. 720 nm illumination was performed with a tungsten lamp through a 720 nm cut-off filter (4 µE m-2 s-1 light intensity). 2.3. P700+ re-reduction kinetics The redox state of P700 was monitored by measuring the absorption changes at 820 nm using a dual-wavelength detector Walz ED-P700DW-E attached to a PAM-102 fluorimeter (Walz, Germany). Measurements were performed at 20°C by placing a light guide close to a cell colony in agar Petri dishes. Each colony was illuminated for 30 s by a FR light from a Walz 101-FR LED at setting 10 in order to oxidize P700. Re-reduction of P700+ in the dark was then recorded. Decompositions of re-reduction kinetics into exponentials were done with Sigmaplot 8.0 software. 2.4. Fluorescence measurements Dark-adapted cell suspensions were successively illuminated by red light (PSII light) for 12 min, red light plus far-red light (PSI light) for 12 min, and, finally, by red light alone. Red light (80 µE m-2 s-1) was provided by a tungsten halogen lamp through a 650 nm cut-off filter. Far-red light was provided by a FR-101 Walz LED connected to a PAM-102 unit, generally set at intensity 10. Maximum fluorescence yield (Fm) was measured during exposure of cell suspensions to a saturating flash (1 s, 3000 µE m-2 s-1) using a pulse amplitude modulation 14/02/2007 7 fluorimeter (Walz, Germany) as described elsewhere [25]. The relative change in fluorescence was calculated as [(Fm1-Fm2)/Fm1)] x 100. For fluorescence spectra, cells from 21-day-old cultures were suspended in 50 mM MESNaOH, pH 6.5, placed in a 3-mm quartz tube and illuminated at 650 nm or 720 nm for 20 min at room temperature with a 1000 W ORIEL 66187 tungsten halogen lamp. Control samples were not exposed to any selective light treatment. All samples were subsequently frozen in liquid nitrogen. Fluorescence spectra were recorded at 77 K by exciting samples with the tungsten halogen lamp and a double 0.22 m SPEX 1680B monochromator. Excitation light was at 470 nm (3.6 nm slit width) and emission was detected from 650 to 800 nm (1.92 nm slit width). Fluorescence was detected through a 0.5 JARREL-ASH monochromator with a Hamamatsu R928 photomultiplier tube. All measurements were corrected from the system response. Chlorophyll fluorescence emission spectra exhibited peaks at 680 nm and 685 nm due to chlorophyll a associated to PSII and at 735 nm from chlorophyll a molecules associated mainly to PSI. All spectra were normalized at 685 nm. 3. Results 3.1. Thermoluminescence emission bands from Photosystem II The thermoluminescence emission technique consists in illuminating a photosynthetic sample at a temperature low enough to block charge recombination processes, then revealing the different types of charge pairs as luminescence emission bands by progressive warming (for a review see [22]). Several TL bands originating from different recombination reactions in PSII can be identified and characterized by this method. In this work, we have studied some of the most relevant TL bands in dark adapted soybean cell suspensions from WT and STR7 cell lines (Fig. 1). Excitation of WT and STR7 cell suspensions with a series of saturating single turn-over flashes at 1ºC induced the appearance of very complex TL glow curves. The light emission curves obtained after 14/02/2007 8 illumination with two flashes were the greatest of the series and are shown in Fig. 1A. These TL signals could be well simulated by two decomposition components with different tmax and contributions to the total signal intensity. We assigned the first component to the well-known TL B band originating from the recombination reactions of S3QBand S2QBcharge pairs. We tentatively assigned the second TL component appearing at higher temperatures to the socalled afterglow (AG) TL emission band usually induced by FR illumination [10]. A similar AG band has been observed in pea, Arabidopsis and tobacco leaves excited by white light [10,26,27]. The mathematical analysis of these two components allowed us to estimate tmax and contribution of B and AG bands to the total signal intensity in WT and STR7 cell lines, respectively. For the B band, we obtained tmax values of about 27ºC for both WT and STR7, and signal contributions of 22% (WT) and 28% (STR7). For the AG band, tmax values of 46ºC (WT) and 43ºC (STR7) and signal contributions of 78% (WT) and 72% (STR7) were obtained. These results show two effects of the mutation: (1) a downshift of about 3ºC for AG band; (2) a slight decrease in the ratio between the intensities of the AG and B bands (from 3.5 in WT to 2.6 in STR7). Analysis of TL yield in dark-adapted samples illuminated by a train of short saturating flashes allows the estimation of the ratio S0:S1 and QB:QBin PSII [22,23]. These experiments showed no differences between WT and STR7 cell lines (data not shown), both of them exhibiting a typical four-oscillation period with maxima after the 2nd and 6th flashes for B and AG bands and ratios of 25:75% and 50:50% for S0:S1 and QB:QB-, respectively. AG band induced by illumination with white light flashes totally disappeared after incubation of both type of cells (WT and STR7) with 10 µM antimycin (Fig. 1A, inset), a well-known inhibitor of the FQR pathway of cyclic electron transport around PSI [2]. This result suggests that most of electrons leading for AG emission in soybean cells are transferred from the stroma to QB via FQR pathway. 14/02/2007 9 To confirm that the proposed AG band observed in soybean cell suspensions after white light flash excitation can be identified as a typical AG band (normally induced by FR light), we have also performed TL measurements after continuous illumination of cell samples with 720 nm (FR) monochromatic light (Fig. 1B). After this illumination, we also excited samples with two white light flashes to ensure induction of maximal signals for B and AG bands. FR illumination generated a more prominent AG band in WT cells, while the B band was reduced. However, for STR7 cells, the intensity pattern was similar to that observed in white light excitation experiments (Fig. 1A). FR light preferentially excites PSI and consequently oxidizes the plastoquinone pool, thus favouring in the dark a back transfer of electrons from stromal reductants to the oxidized QB and finally to the S2 and S3 states of the manganese cluster [9]. This overall recombination reaction leads to AG emission. Interestingly, FR excitation does not increase the amount of AG band in STR7 mutant. The mathematical analysis of the two components found by the simulation software showed a slight upshift for the B band (tmax of 26ºC for WT and 28ºC for STR7), a significant downshift of about 4ºC for the AG band (tmax of 46ºC for WT and 42º C for STR7) and an important decrease in the ratio between the intensities of AG and B bands from 4.5 (WT) to 2.3 (STR7). These results support that the 46ºC-band observed in soybean cell suspensions after excitation with white light flashes corresponds to the same recombination reaction which gives rise to the FRinduced AG TL band described in [10]. They also confirm the AG band downshift observed in STR7 TL curves generated by white light flashes (Fig. 1A). A similar AG band downshift has been detected in maize leaves when PSI cyclic electron flow is induced [8]. In order to study recombination reactions involving QA, TL band emissions obtained after flashing WT and STR7 soybean cell suspensions previously incubated in the presence of 50 µM DCMU have been recorded (Fig. 1C). In these samples after one flash of white light, the B and AG bands completely disappeared and two other TL bands were detected: (1) at low temperatures, a well-known Q band corresponding to the S2QArecombination; and (2), at 14/02/2007 16 not suggest any reason why LHCII could not be phosphorylated or dephosphorylated in the mutant. Thus, the only possible explanation to understand why the state transitions are blocked in the mutant is that there is no movement of the dephosphorylated LHCII from PSI towards PSII. As already mentioned in this paper, the thylakoid membrane of the STR7 mutant is more rigid than that of the WT [14, 16]. An increase in the thylakoid membrane rigidity could be responsible for locking the STR7 cells in state 2 due to impaired lateral diffusion of protein complexes. A similar conclusion was obtained when state transitions were studied in cyanobacterial mutants defective in thylakoid membrane unsaturation [41]. The analysis of Arabidopsis mutants defective in thylakoid membrane unsaturation, lacking mutations in the psbA gene [42], may help to clarify this mechanism. It is more difficult to understand why a point mutation in the PSII D1 protein induces such changes. The plant ω3 fatty-acid desaturases FAD7, FAD8 and FAD3 are responsible for the production of trienoic fatty-acid. Their activities are responsible for the maintenance of appropriate thylakoid membrane fluidity. The expression of these enzymes seems to be regulated in response to light [43] and to photosynthetic electron transport activity (Collados, Picorel and Alfonso, unpublished observations) in soybean cells. Studies are in progress in our laboratory to further test this hypothesis. Acknowledgements This work was supported by grants from the Ministry of Education and Culture of Spain (BFU-BMC2004-04914-C02-01, BMC2002-00031 and BFU-BMC2005-07422-C02-01) and Andalusia Government (PAI CVI-261). The authors thank Dr. J.R. Pérez-Castiñeira and Dr. A.M. Lindahl for critical reading of the manuscript. We also thank M.V. Ramiro for helpful technical assistance and Dr. R. Cases (ICMA-CSIC) for help in fluorescence spectra measurements. 14/02/2007 17 References [1] N. Bukhov, R. Carpentier, Alternative photosystem I-driven electron transport routes: mechanisms and functions, Photosynth. Res. 82 (2004) 17-33. [2] D.S. Bendall, R.S. 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Andreu, R. Picorel, M. Alfonso, A light-sensitive mechanism differently regulates transcription and transcript stability of omega3 fatty-acid desaturases (FAD3, FAD7 and FAD8) in soybean photosynthetic cell suspensions, FEBS Lett. 580 (2006) 4934-4940. 14/02/2007 22 Figure legends Figure 1. (A) Thermoluminescence glow curves from soybean cells of WT and STR7 lines recorded after two flashes. The dashed lines represent the simulation components corresponding to the best fit (see Materials and Methods). Inset: TL signal recorded after two flashes from WT and STR7 soybean cells incubated in the presence of antimycin. Darkadapted cell suspensions were incubated for 5 min in the dark at room temperature with 10 µM antimycin. (B) Thermoluminescence glow curves from WT and STR7 mutant soybean cells recorded after two flashes with previous illumination with 720 nm monochromatic light. Cells resuspended in 50 mM Mes-NaOH, pH 6.5 buffer were dark-adapted for 2 min at 20ºC and then irradiated 5 min with 720 nm light at 20ºC, and subsequently cooled to 0ºC during 1 min. Two flashes were given at the end of this period. The dashed lines represent the simulation components corresponding to the best fit (see Materials and Methods). (C) Thermoluminescence glow curves after one flash of WT and STR7 cells previously incubated in the presence of DCMU. Dark-adapted cell suspensions were incubated in darkness at room temperature with 50 µM DCMU. Figure 2. HTL measurements recorded from 10ºC to 160ºC from WT and STR7 mutant soybean cells. Only the 70-160ºC range of the HTL bands is shown. Samples were darkadapted for 1 min at 10ºC and heated with a slow warming rate of 0.1 ºC/s from 10ºC to 160ºC without flash excitation. In order to desiccate the samples and prevent any oxidation induced by high temperature, N2 was flushed on the samples during the HTL experiments. Figure 3. Dark re-reduction of P700+ in soybean cells of WT and STR7 mutant. P700+ reduction was measured by monitoring absorbance changes at 830 nm in the dark after oxidation induced by a 30-s pulse of FR light. The measurements were made directly on 14/02/2007 23 single colonies in agar Petri plates at 20ºC. Experimental data are shown as open circles and the lines represent the biexponential fitted curves. Figure 4. (A) Measurements of transitions between state 1 and state 2 in WT and STR7 mutant soybean cells. Dark-adapted cells were exposed either to light favouring PSII excitation (650 nm monochromatic light) or light favouring PSI excitation (720 nm monochromatic light). The fluorescence decreases when illumination includes PSI light and increases again when illumination is changed to favour PSII. (B) 77 K fluorescence emission spectra of WT and STR7 mutant cells incubated under light 2 (Solid line) and light 1 (dotted lines) for 20 min immediately prior to freezing at 77 K. Light 2, 650 nm monochromatic light; Light 1, 720 nm monochromatic light. All spectra were normalized at 685 nm. Table 1. Kinetic parameters of P700+ dark reduction in soybean STR7 mutant and WT cells. Fast phase Slow phase Amplitude (%) t ½ (s) t ½ (s) WT 90 ± 4 1.52 ± 0.07 3.35 ± 0.50 STR7 89 ± 5 0.45 ± 0.06 3.11 ± 1.01 Experimental conditions were described in Materials and Methods. Amplitudes and half-lives were calculated according to a biexponential curve fit. Data are the means ± SD of five independent experiments. Table 2. Measurements from 77 K fluorescence emission spectra of soybean STR7 mutant and WT cells. Mean ± SD, n = 3 Light treatment F685/F7381 F685/F738 WT STR7 Light adapted2 0.29 ± 0.008 0.31 ± 0.005 650 nm 0.28 ± 0.007 0.31 ± 0.006 720 nm 0.38 ± 0.009 0.29 ± 0.005 State transition3 26% -7% 1F685: fluorescence at 685 nm; F738: fluorescence at 738 nm 2 (10 μΕ m-2 s-1) 3State transition: [(F685/738(ox)–F685/738(red))/F685/738(ox)] x 100. F685/738(ox) = fluorescence ratio obtained under illumination with 720 nm light; F685/738(red) = fluorescence ratio obtained under illumination with 650 nm light