FIRST INDUCED PLASTID GENOME MUTATIONS IN AN ALGA WITH SECONDARY PLASTIDS: psbA MUTATIONS IN THE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) REVEAL CONSEQUENCES ON THE REGULATION OF PHOTOSYNTHESIS1
3 5 2 Arne C. Materna ,5, Sabine Sturm , Peter C. Kroth, andJohann Lavaud ,4 Group of Plant Ecophysiology, Biology Department, Mailbox M6ll, University of Konstanz, Universitatsstraf3e 10, 78457 Konstanz, Germany
Diatoms play a crucial role in the biochemistry RC, photosystem 11 reaction center; QA and QB, and ecology of most aquatic ecosystems, especially quinone A and B; WT, wildtype because of their high photosynthetic productivity. They often have to cope with a fluctuating light climate and a punctuated exposure to excess light, Diatoms (Heterokontophyta, Bacillariophyceae) which can be harmful for photosynthesis. To gain are a mqjor group of microalgae ubiquitous in all insight into the regulation of photosynthesis in marine and freshwater ecosystems. With probably diatoms, we generated and studied mutants of the >10,000 species, their biodiversity is among the diatom Phaeodactylum tricornutum Bohlin carrying largest of photosynthetic organisms, just after the functionally altered versions of the plastidic psbA higher plants (Mann 1999). Diatoms are assumed gene encoding the D 1 protein of the PSII reaction to contribute to about 40% of the aquatic primary center (PSII RC). All analyzed mutants feature an production (i.e., ",20% of the annual global pro amino acid substitution in the vicinity of the QB duction) and to play a central role in the biochemi binding pocket of Dl. We characterized the photo cal cycles of silica (which is part of their cell wall) synthetic capacity of the mutants in comparison to and nitrogen (Sarthou et al. 2005). Their produ wildtype cells, focusing on the way they regulate ctivity has contributed largely to the structure of their photochemistry as a function of light intensity. contemporary aquatic ecosystems (Falkowski et al. The results show that the mutations resulted in 2004). In contrast to the supposed primary origin constitutive changes of PSII electron transport rates. of red algae, green algae, and higher plants, dia The extent of the impairment varies between toms originate from a secondary endosymbiotic mutants depending on the proximity of the muta event in which a nonphotosynthetic eukaryote prob tion to the QB-binding pocket and/or to the non ably engulfed a eukaryotic photosynthetic cell heme iron within the PSII RC. The effects of the related to red algae and transformed it into a plas mutations described here for P. tricornutum are simi tid (Keeling 2004). This peculiar evolution has lar to effects in cyanobacteria and green microalgae, led to complex cellular functions and metabolic emphasizing the conservation of the DI protein regulations recently highlighted by the publication structure among photosynthetic organisms of differ of the genome of two diatom species (Armbrust ent evolutionary origins. et al. 2004, Bowler et al. 2008); Thalassiosira Key index words: chlorophyll fluorescence; D 1 pseudonana and Phaeodactylum tricornutum. The com protein; diatom; electron transport; herbicide; plex cellular functions include aspects of photosyn photosystem 11; QB pocket thesis (Wilhelm et al. 2006), photoacclimation (Lavaud 2007), carbon and nitrogen metabolism Abbreviations: DCMU, (3-(3, 4-diclorophenyl)-I, (Alien et al. 2006, Kroth et al. 2008), and response I-dimethylurea); LHC, light-harvesting complex; to nutrient starvation (Alien et al. 2008). OEC, oxygen evolving complex; PAM, pulse As for most microalgae, the photosynthetic effi amplitude modulation; PQ, plastoquinone; PSII ciency and productivity of diatoms strongly depend on the underwater light climate (MacIntyre et al. 2000). Planktonic as well as benthic diatoms tend I Received 27 June 2008. Accepted 16 March 2009. to dominate ecosystems characterized by highly 2Author for correspondence: e-mail [email protected]. turbulent water bodies (coasts and estuaries) where "Present address: Aim Laboratory, Civil and Environmental Engi- they have to cope with an underwater light climate neering, Massachusetts Institute of Technology (MIT), 77 Massachusetts Ave., 48-208, Cambridge, MA02139, USA. with high-frequency irradiance fluctuations coupled 1Present address: UMR CNRS 6250 'LIENSs', Institute for Coastal with large amplitudes. Depending on the rate of and Environmental Research, University of La Rochelle, 2 rue water mixing, diatoms can be exposed to punctual Olympe de Gouges, 17042 La Rochelle Cedex, France. or chronic excess light, possibly generating stressful "These authors contributed equally to this work.
838 PHOTOSYNTHESIS IN psbA (Dl) DIATOM MUTANTS 839 conditions that impair photosynthesis (i.e., Cell cultivation and preparation for physiological measw·ements. photoinactivation/-inhibition) (Long et al. 1994, P. tricomutum WT and mutant cells were grown in 200 mL Lavaud 2007). In higher plants and cyanobacteria, sterile F/2 50% medium (Guillard and Ryther 1962) at 21°C in airlifL columns continuously flushed with sterile air. The the processes of PSII RC photoinactivationl cultures were illuminated at a light intensity of 50 J.lmol 2 -inhibition are strongly influenced by the redox photons' m- • S-I with white fluorescent tubes (L58W/25, state of the acceptor side of PSII with quinones Unive~'sal white, OSRAM GmbH, Munich,' Germany) with a (QA and Qn) as primary electron acceptor (Vass 16:8 hght:dark (L:D) cycle. Cells were harvested during the et al. 1992, Fufezan et al. 2007). exponential phase of growth, centtifuged (Allegra 25R; Beck Here we report on the generation and character man Coulter GmbH, Krefeld, Germany) at 3,OOOg for 10 min, and resuspended in their culture medium to a final chI ization of four psbA mutants of P. tricomutum. All I a concentration of 10 ~tg chI a' mL- . The algae were mutants feature distinct amino acid exchanges in continuously stirred at 2JOC under low continuous light. For the Dl protein of PSII close to or within the oxygen (02), chI fluorescence, and thennoluminescence Qn-binding pocket. The point mutations resulted in measurements, cells were dark-adapted 20 min prior to a constitutive impairment of the PSII electron trans measurement. fer in all mutants to different extents. To our knowl Protein extraction and Westem blot analysis. Cells were har edge, this is the first report of plastid genome vested during the exponential phase of growth as described above and subsequently grinded in liquid nitrogen. The mutants in an alga with secondary plastids. homogenized cells were resuspended in preheated (60°C) extraction buffer (125 mM Tris/HCI [pH 6.8], 4% [w/v] SDS, MATERIALS AND METHODS 200 ~tM PMSF, and 100 mM DTT) and, after heat treatment extracted with acetone. After wash steps; the dried Strains and media used for producing the psbA mutants of protei~ pellet was finally resuspended in extraction buffer. Total P. tricornutum. P. tricomutum (University of Texas Culture protein was separated by SDS-PAGE. Proteins were transferred Collection, strain 646) was grown at 22°C under continuous 2 electrophoretically onto a PVDF membrane (Hybond"'-P, illumination at 50 ~tmol photons' m- . S-I in Provasoli's Amersham Biosciences UK Limited, Buckinghamshire, UK) enriched F/2 seawater medium (Guillard and Ryther 1962) an~ incubated with an an~iserum against Dl (Anti-PsbA global using 'Tropic Marin' artificial seawater at a final concentration antibody, ~S05 084, Agnser~, Sweden). Detection was per of 50%, compared to natural seawater. When used, solid media formed usmg the chemolummescence detection system from contained l.2% Bacto Agar (Difco Lab., Becton Dickinson and Roche Diagnostics (BM Chemiluminescence Blotting Substrate Co., Sparks, MD, USA). POD; Roche Diagnostics GmbH, Mannheim, Germany). Generation ofpsbA mutants from P. tricornutum. Construction Pigment extraction and analysis. ChI a amount was deter of plasmid transformation vectors: Four transformation vectors mined by spectrophotometry using the 90% acetone extrac were con~t~cted harboring a 795 bp psbA fragment containing tion method. For pigment extraction, cells were deposited on the Qs-bmdmg pocket. The psbA inserts of each vector carried a filter and frozen in liquid nitrogen. Pigments were extracted individual point mutations leading to different substitutions of with a methanol:acetone (70:30, v/v) solution. Pigment the amino acid serine encoded by the PsbA (Dl) codon 264 analysis was performed via HPLC as previously described (for d~tails and a.v~ctor map, see Fig. SI in the supplementary (Lavaud et al. 2003). Cell counts were performed using a materIal). In addition to the nonsynonymous point mutations Thoma hematocytometer (LaborOptik, Friedrichsdorf, in codon 264, a second, silent point mutation was introduced Germany). . into codon 268 (TCT to TCA, nt position 804) without Spectroscopy and PSI reaction center (P700) concentration. The ch~nging th~ encoded amino acid. The purpose of the second absorption spectra were obtained at room temperature with a pomt mutatIon was to delete a BssSl restriction site, thus DW-2 Aminco (American Instrument Co., Jessup, MD, USA) allowing easy RFLP screening of putative transformants. spectrophotometer, half-bandwith 3 nm, speed 2 nm . s-I, Biolistic transformation ofP. tricornutum: Transformation of OD = 0 at 750 nm, 50% F/2 medium as a reference. P P. tricomutum was performed using a Bio Rad Biolistic PDS 700 quantity relative to chI a was determined as described earlier lOOO/He Particle Delivery System (Bio-Rad, Hercules, CA, (Lav~ud et al. 2002a) with the DW-2 Aminco spectrophotom USA~ as described previously (Kroth 2007). Gold particles with eter m dual beam mode (reference at 730 nm). a diameter of 0.1 ~tm served as microcarriers for the Thermoluminescence. Thermoluminescence patterns were DNA constructs. Bombarded cells were allowed to recover for measured with a self-made thermoluminometer following the 24 h before being suspended in 1 mL of sterile F/2 50% procedure previously described (Gilbert et al. 2004). Flashes medium. Transformants (250 ~tL) were selected at 21°C under 2 were single turn-over with duration of 25 ~ts. Samples were constant illumination (35Jtmol photons' m- . S-I) on ag'ar adjusted to 20 ~tg chI a' mL -I for measurement. plates containing 5 lO-b M DCMU herbicide (3-(3, 4-di Oxygen (0 ) concentration and photosynthetic light-response (P/E) clorophenyl)-I,I-dimethylurea) and repeatedly streaked on 2 cumes. O concentration was measured with a DWI-Clark fresh ~olid selective medium to obtain full segregation of the 2 elect~ode (H~nsate~h Ltd., Norfolk, UK) at 21°C. White light mutatIOn. of adjustable mtenslty (measured with a PAR-sensor, LI-185A; Isolation of DNA and sequencing of wildtype and mutant psbA Li-Corlnc., Lincoln, NE, USA) was provided by a KL-1500 genes. Total nucleic acids from the wildtype (Wf) and mutant quartz iodine lamp (Schott, Mainz, Germany). Cell culture cells were isolated via a cetyltrimethylammoniumbromide samples were dark-acclimated for 20 min before measurement. (CTAB)-based method (Doyle and Doyle 1990). Prior to the P/l~ curves. were ?btail~ed ~y ill~I?inating a 2 mL sample mutagen~sis of P. tricomutum, the WT psbA gene and the dunng 5 mm at vanous hght mtensltIes. A new sample was used surroundmg genes were sequenced (NCBI accession no. [or each measurement. El{, the irradiance for saturation o[ AY864816) via primer walking (GATC, Konstanz, Germany). photosynthetic O emission was estimated from PIE cUlves. For the molecular characterization of mutants, a 795 bp 2 Chi fluorescence induction hinetics and DCMU resistance. ChI a fragment of the jJsbA gene was amplified as described in fluorescence induction kinetics were performed with two Figure SI, and both strands were fully sequenced (GATC, instrumen.ts: a ~)E"':-f1uorometer (Walz, Effeltrich, Germany) Konstanz, Germany). [or short-Ume kmeucs (up to 200 ms), which allowed a classic 840 ARNE C. MATERNA ET AL.
OIJP analysis (see Fig. S4 in the supplementary materials for same pair of point mutations that was supposed to be details), and a self~made "continuous light" fluorometer introduced into psbA by the utilized transformation (Paresys et al. 2005) for long-time kinetics (up to 100 s). Cells vector (data not shown). Negative control experi were adjusted to a concentration of 5 ~lg chi a' mL-\ and ments involving exclusive selection without preceding 20 ~lg chi a' L -\ for the PEA- and the sel1~made fluorometers, respectively. transformation, and biolistic transformation without DCMU resistance was evaluated measuring the inhibition of vector DNA failed to generate resistant colonies. We the PSII activity versus increasing DCMU concentrations (see sequenced 1,000 bp regions surrounding the ~ Fig. S4 for details). The kill curves with DCMU and atrazine pocket as well as coding and noncoding areas more were performed by growing the cells on solid medium with distant to the psbA locus without finding other muta increasing concentrations of herbicides; growth conditions tions than the ones described here. Yet, we cannot were the same as described before. ChI fluorescence yield. Chi fluorescence yield was monitored exclude the possibility that additional mutations have with a modified PAM-IOl fluorometer (Walz) as described occurred at unknown loci. However, due to the selec previously (Lavaud et al. 2002a). For each experiment, 2 mL tion on DCMU, which specifically interacts with the was used. Sodium bicarbonate was added at a concentration of ~ pocket of the D1 protein, additional mutations at 4 mM to prevent any limitation of the photosynthetic rate by other loci are likely to be detrimental and therefore carbon supply. When used, DCMU was incubated with the cell selected against. suspension at the beginning of the dark-adaptation period. Fluorescence parameters were defined as described in Although intriguing, this study is not focusing on Figure S4. The parameter used to estimate the fraction of the underlying molecular mechanism leading to the reduced ~ (Buchel and Wilhelm 1993) was 1 - qP where qP elevated mutation rates in the psbA gene; it will be is the photochemical quenching of chi fluorescence. The rate the focus of a subsequent work. Instead, we charac of linear electron transport was calculated as follows: terized and compared in four selected mutants the physiological effects of different amino acid substitu ETR = <1>PSII x PDF x C( x 0.5 (1 ) tions in the D 1 protein of the PSII RC on the regu where <1>PSII is the PSII quantum yield for photochemistry, lation of photosynthesis. PDF is the irradiance, and L275W WT kDa higher J phase (+23% and 57%, respectively). S264A showed a drastically increased (by 71 %) and delayed J phase and, in contrast to the other mutants, an D1 83 increased I phase (see inset Fig. 2C and Fig. S4). cross-links When recorded over a longer timescale (lOO s) and at continuous illumination, the pattern of the fluo 01 32.5 rescence induction kinetics of S264A and L275W was different (only L275W is shown, Fig. 2D). In 25 S264A and L275W, the amplitude of the 1-45 ms peak increased, and the whole pattern of the kinet 23kDa - ics was disturbed. The Fo chI a fluorescence level was increased in FIG. 1. Western blot of the D1 protein of the PSII reaction all mutants (Fig. S4). Adding DCMU (resulting in center of Phaeodactylu1I! tricornutu1I! wild type (WT) and the psbA inhibition of electron transport between ~ and mutant L275W cells. Cells were grown at 50 J.Lmol photons' 2 Qs) to WT cells resulted in an increased Pi) m- . S-I. Bands representing D1 degradation products of 23 kDa and the cross-link products of ~83 kDa also resulting from D1 (195 ± 6.5) comparable to S264A and L275W. When degradation (Ishikawa et al. 1999) were found in a larger amount gro,wn at low light intensity (50 /lmol photons . 2 in L275W but not in the WT. m- • S-1) all mutants showed a maximum photosyn thetic efficiency of PSII (F,/Fm' Fig. S4), which was similar to the WT cells, except for L275W (-19%). 2 intensity (50 /lmol photons' m- • S-1), the pigment When measured at an equivalent irradiance, the contents of all the mutants and the WT cells were effective PSII quantum yield (PSII, Fig. S4) was very similar, although the mutants tended to accu the same for WT cells and V2191, but lower in the mulate slightly more chI a per cell (see Fig. S4). other mutants. These values were in accordance The concentrations of active PSII RCs per chI a; with the steady-state electron transport rate per PSII 1::<;s, and RClCSo, were higher in all the mutants but (ETo/CSo, Fig. S4). Addition of DCMU to the WT L275W (Fig. S4). The low concentration for L275W resulted in a decreased A B 700 -.-wr Q) 600 --o--V2191 0 -<>- F2551 :J c: Q) 500 """S264A ~ 1.5 0 I/) Q) Q) Cl c: 400 c: (\l 'E .c .2 300 o Q 1: ....E 200 Q) Q) t: 0.5 .c :J I- 100 b ...D. ••• D. ••• t:;,.. o 0 0 10 20 30 40 50 5 10 15 20 Temperature ("C) Flash number, n C D 400 ---. :J 0.8 ::i ~ ~300 Q) Q) 0 0.6 o c: c: Q) 0 ~ 200 I/) 0.4 I/) Q).... Q).... Q Q :J .2 100 u:: 0.2 u. 0 0.01 0.1 1 10 100 Time (s) FIG. 2. (A) Thermoluminescence emission of dark-adapted cells of Phaeodactylum triwl'nutum wild type (WT) and the psbA mutants V219I/ F255I / S264A. The characteristic emission bands at 7°C (Q) and 22°C (B) are shown; they reflect the recombination states of the PSII reaction center S2~ - and S2/3Qn - and the redox potential of ~ and Qn, respectively (Gilbert et al. 2004, Eisenstadt et al. 2008). Curves represent the average of three measurements. (B) O 2 production in a sedes of single-tumover flashes (02 sequences) by dark-adapted cells of P. tricornutum WT and of the two psbA mutants S264A and L~75W, as measured via a flash electrode. The pattem of the O2 sequence for V219I resembled the one of the WT, and the pattern of F255I resembled the one of S264A with less pronounced fea tures. See Figure S4 (in the supplementary material) for a detailed description. (C-D) Chi a fluorescence-induction kinetics reflect quan tum yield changes of chi a fluorescence as a function of the illumination duration, which relates to both excitation trapping in PSII and the ensuing photosynthetic electron transport. (C) Short-time kinetics recorded via PEA fluorometer from dark-adapted cells of P. tricornutum WT and the four psbA mutants (V2191 / F2551 / S264A / L275W). The letters 0, j, I, P, H, and G refer to the phases of the kinetics (Lazar 2006). (D) Long-time kinetics from dark-adapted cells of WT and L275W (same pattern for S264A) as recorded with a self~made "continuous light" fluorometer. The arrow indicates the first peak (I phase at 45 ms). The amplitude of I reflects the redox state of ~ (Lavaud and Kroth 2006). In diatoms, the classic P peak is divided into two peaks, Hand G (Lavaud and Kroth 2006, Lazar 2006). L275W at a light intensity of 250 Ilmol photons . reduction state of ~ (+ 10%) and the decreased 2 m- . S-I; at this light intensity, the extent of ~ ETR per PSII (about 5%), in V2191 the number of reduction was close to its maximum (Fig. 3A). PSII RCs was increased (14% to 22%, depending on the method) to maintain a photosynthetic activity similar to the WT as reflected also by its growth DISCUSSION pattern. Hence, the exchange of Val to lie at Three out of the four psbA mutants showed a the position 219 in the helix D appears to be too phenotype clearly distinct from the WT (see Fig. 4). distant from the Qs-binding pocket to significantly Obviously, the observed amino acid substitutions disturb the electron transport within the PSII RC in hold implications for the phenotype of the mutants. P. tricomutum. The phenotypic effects described in this study allow Effects of mutations within the 0rbinding pocket: F2551 various insights into the functionality of mutated and S264A. The residues Phe255 and Ser264 bind residues or domains within the Dl protein. the head group of Qs (Kern and Renger 2007). A mutation that slightly affects the photosynthetic The electron transport between ~ and Qs in F2551 efficiency: V219I. In response to the slightly increased was significantly impaired (Fig. 4), slowing down the PHOTOSYNTHESIS IN psbA (Dl) DIATOM MUTANTS 843 A B 35 1_----- 30 0.8 ,1, ...... ~ ...... ,;.;Q; ... -- ,...... l{.. - ,,*:,:,,;e 'en I' ...... 25 . j" C)I 0.6 E k... -----O------~ 0- .1 I ,,' 20 C" I ' '0 ...-I E 0.4 ..::: 15 'f}' r•••..,.,...... -8' . .-:"".....- .-:-;,.. ":":" .. -:-:,~ 1 0::: I \Y' r __-- --' I- 10 WT ill 1/ --o--V2191 0.2 ooO-F2551 5 V2191 ""'''S264A 20 40 60 60 100 . O-L275W Oc~--~--~----~--~----w o 200 400 600 800 1000 0~0----~50~0~--1~0700~~1~50~0~~2~000 Light intensity (IJmol photons· m·2 . S·1) Light intensity (IJmol photons· m·2 . S·1) FIG. 3. Chi a fluorescence parameters as recorded with a PAM·fluorometer for the wildtype (WT) and the four jJsbA mutants (V219I / F255I / S264A / L275W) of Phaeodactylurn tricomuturn cells as a function of a li~ht intensity gradient from darkness (0 !lmol photons· 2 m- . S-I) to the equivalent of full sunlight in nature (2,000 !lmol photons' m- . S-I, Long et al. 1994). The illumination duration was 5 mini a new sample was used for each irradiance treatment. (A) 1 - qP estimates the fraction of reduced ~ (Biichel and Wilhelm 2 1993). Inset: Ratio mutants versus WT of the amplitude of the 1-45 ms peak (see Fig. 2D) up to 100 !lmol photons' m- • S-I. (B) ETR is the rate of linear electron transport. See the Materials and Methods section and Figure S4 (in the supplementary materials) tor details about the calculations of these parameters. Values are average ± SO of three to tour measurements. reoxidation of ~ - as illustrated by the increased changes, the potential for photochemistry, qP, was ~ Qn -I~ -Qn- state. It was especially visible with decreased at interm~dia~ irradiances (up to 2 the pattern of thermoluminescence that resembles 400 J..lmol photons' m- . s- ). At high light intensi the one reported in P. tricomutum for WT cells trea· ties, the ETR was reduced (Fig. 4), reflecting the ted with DCMU (abolishment of the B band and decrease of the PSI! antenna size and of WT QA~conc. OEC Active QS-toQA EiRma• PSI! El{ PSI:PSI! back-transfer PSI! RCs antenna size V2191 not ...... disturbed (+ 10%) (-5%) (+14%) disturbed .... (+23%) (+ 6%) (-15%) (+ 51%) (- 22%) (+ 20%) highly .... (+71%) (+30%) disturbed (_ 50%) (+66%) (-40%) (+31%) L275W highly (+57%) (+37%) disturbed (_ 51%) (-52%) (-31%) (+26%) (+27%) FIG. 4. Diagram of the influence of mutations on the photosynthetic apparatus of the psbA mutants (V219I1F255I1S264A1L275W) of Phaeodactylu1I! tricornutwn in comparison to the wildtype (WT) situation. Left: the electron pathways within the PSII reaction center; right: the architecture of the photosystems as a function of WT situation (PSI:PSII 1 :2); arrow up: increased value (the true value is given in-between brackets); arrow down: decrease; flat arrow: no change. Symbols: red star, mutation; size of QA-/Qa-, concentration of QA -lQa-; thickness of the e - arrows, value of the ETR"m, and of the Qa - to QA back-transfer; dotted feature of the OEC arrow, propor tion of the disturbance of the OEC operation. El{, light intensity for saturation of photosynthesis; e-, electrons; LHC, light-harvesting antenna complex; OEC, oxygen evolving complex; PSIIPSII RC, PSII/PSI reaction center; PSI:PSII, molar photosystem stoichiometry; QA and Qa, quinones. See the text for a more detailed description. The effect of the L275W mutation on the photosyn ported by the high Fo level and the lowest F./Fm • In thetic ability per PSII was similar to the point muta contrast to the other mutants, L275W responded to tion S264A (Fig. 4) but showed a highly disturbed the point mutation by modifying the architecture of OEC operation along with increased ~ reduction. the photosynthetic apparatus as illustrated by the ~ reduction was already elevated (1.2- to 1.3-fold increase of the PSI:PSII stochiometry (Fig. 4). This compared to WT) at light intensities that were even attempt to maintain a reasonable photosynthetic below the intensity used for growing the cells. Ulti activity might lead to an increased capacity for PSI mately, L275W showed a decreased growth rate cyclic electron flow as in higher plants with deficient under low light as well as the inability to reach the linear electron transport (Kotakis et al. 2006). same final maximal biomass. The main difference In a series of papers (reviewed in van Rensen compared to the other mutants was the reduced et al. 1999), Govindjee et al. showed that the amount of active PSII (Fig. 4), demonstrated by a exchange of the residue Leu275 significantly per disturbed D1 repair cycle. Mutations close to or turbs the ~-Fe-Qs structure, the protonation of within the Qn pocket have been reported to modify Qn2- (Xiong et al. 1997), and subsequently the PQ the D1 turnover either by accelerating its damage redox state. It is thus likely that the phenotype of and/or by inhibiting its proteolysis and/or syntheSis L275W is due to the close vicinity of the point muta (Della Chiesa et al. 1997, Nishiyama et al. 2006). tion to both the Qn pocket and the nonheme iron Thus, it is very likely that in L275W there is a mixed atom binding sites, which functionally affects the population of active and inactive PSII, even at low properties of both the ~ and Qn pockets (Vermaas light intensities (Mohanty et al. 2007), which is sup- et al. 1994). PHOTOSYNTHESIS IN psbA (Dl) DIATOM MUTANTS 845 Effects of similar mutations in cyanobacteria and green Biichel, C. & Wilhelm, C. 1993. In vivo analysis of slow chlorophyll algae. The effects of the mutations described here fluorescence induction kinetics in algae: progress, problems and perspectives. Plwtochem. Photobiol. 58: 137-48. for the diatom P. tricornutum are similar to effects of Della Chiesa, M., Friso, G., Deak, Z., Vass, I., Barber,.J. & Nixon, P. the same mutations reported in other photosyn 1997. Reduced turnover of the Dl polypeptide and photo thetic organisms. For example, it has also been con activation of electron transfer in novel herbicide resistant cluded that in the green alga Chlamydomonas mutants of Synechocystis sp. PCC 6803. /<'ur. ]. Biochem. 248: (Erickson etal. 1989), the V2191 amino 731-40. reinhardtii Doyle, j..J. & Doyle,.J. L. 1990. A rapid total DNA preparation acid substitution does not significantly disturb the procedure for fresh plant tissue. Focus 12: 13-5. electron transport within the PSI! RC. Also, the Eisenstadt, D., Ohad, 1., Keren, 1. & Kaplan, A. 2008. Changes in effects of the F255I, S264A, and L275W mutations the photosynthetic reaction center n in the diatom Phaeo have been described in cyanobacteria (Synechocystis dactytum tricomutum result in non-photochemical.f1uorescence quenching. Environ. Microbiol. 10: 1997-2007. and Synechococcus) and C. reinhardtii (Erickson et al. Erickson,.J. M., Pllster, K, Rahirc, M., Togasaki, R. K, MClS, L. & 1989, Etienne et al. 1990, Gleiter et al. 1992, Kless Rochaix, .J.-D. 1989. Molecular and biophysical analysis of et al. 1994, Perewoska et al. 1994). Remarkably, the herbicide-resistant mutants of Chlamydomonas reinhardtii: S264A-induced DCMU resistance was much higher structure-function relationship of the photosystem n D I in P. than in all previously studied polypeptide. Plant Cell 1:361-71. tricornutum Etienne, A.-I.., Ducruet, .J.-M., Ajlani, G. & Vernotte, C. 1990. organisms (Gleiter et al. 1992). Comparative studies on electron transfer in photosystem n of herbicide-resistant mutants from different organisms. Biochim. Bi'1Jhys. Acta 1015:435-40. CONCLUSION Falkowski, P. G., Katz, M. E., Knoll, A. H., Quigg, A., Raven,.J. A., Schofield, O. & Taylor, F. R. .J. 2004. The evolution of modern Our results illustrate that not only the substitution phytoplankton. Science 305:354--60. loci but also the nature of the exchanged amino Fufezan, C., Gross, C. M., Sjiidin, M., Rutherford, A. W., acids are essential in modifying the spatial arrange Krieger-Liszkay, A. & Kirilovsky, D. 2007. Influence of the redox ment and properties of the D1 protein (Kless and potential of the primary quinone electron acceptor on pho Vermaas 1994). Ultimately, such structural changes, toinhibition in photosystem n.]. Biot. Chem.282:12492-502. Gilbert, M., Wagner, H., Weingart, I., Nieber, K, Tauer, G., especially in the Qs-binding pocket, are defining the Bergmann, F., Fischer, H. & Wilhelm, C. 2004. A new type electron transport rate within the PSI! RC (Lardans of thermoluminometer: a highly sensitive tool in applied et al. 1998, Oettmeier 1999). The fact that photo photosynthesis research and plant stress physiology.]. Plant synthesis is impaired at different levels in the Physiol. 161:641-51. Gleiter, H. M., Ohad, N., Kolke, H., Hirschberg,.J., Renger, G. & P. tricornutum psbA mutants described here (see Inoue, Y. 1992. Thermoluminescence and flash-induced .Fig. 4) provides a unique opportunity to further oxygen yield in herbicide resistant mutants of the Dl protein study the regulation of photosynthesis in diatoms. in Synechococcus PCC7942. Biochim. Biophys. Acta 1140:135-43. Guillard, R. R. R. & Ryther,.J. H. 1962. Studies of marine planktonic This work was supported by the European network diatoms. 1. C. nana (Hustedt) and D. confervacea (Cleve) Gran. MarGenes (QLRT-2001-01226 to P. G. K), the University Can.]. Microbiol. 8:229-38. of Konstanz (University of Konstanz, 'Anreizsystem Ishikawa, Y., Nakatani, E., Henmi, T., FeIjani, A., Harada, Y., zur Frauenfiirderung' to S. S. and J. L.), and the DFG (grant Tamura, N. & Yamamoto, Y. 1999. Turnover of the aggregates LA2368/2-1 to J. L.). We thank I. Adamska (University of and cross-linked products of the D I protein generated by Konstanz), C. Bowler (ENS Paris), and C. Wilhelm (Univer acceptor-side photoinhibition of photosystem n. Biochim. sity of Leipzig) for access to some of the instruments used Biophys. Acta 1413:147-58. here and for helpful discussions; D. Ballert for technical Keeling, P. 2004. A brief history of plastids and their hosts. Pmtist 155:3-7. assistance; V. Reiser and P. Huesgen (University of Kern,.J. & Renger, G. 2007. Photosystem n: structure and mecha Konstanz), B. Rousseau (ENS Paris), T. Jakob, and H. Wag . nism of the water: plastoquinone oxidoreductase. Photosynth. ner (University of Leipzig) for help with some of the experi Res. 94:183-202. ments. This work is part of the PhD project of A. C. M. and Kless, H., Oren-Shamir, M., Malkin, S., McIntosh, I.. & Edelman, M. of the diploma work of S. S. 1994. The D-Eregion of the Dl protein is involved in multiple quinone and herbicide interactions in photosystem n. Allen, A. E., LaRoche,j., Maheswari, U., Lommer, M., Schauer, N., Biochemistry 33: 1050 1-7. Lopez, P. j., Finazzi, G., Fernie, A. R. & Bowler, C. 2008. Kless, H. & Vermaas, W. 1994. Many combinations of amino acid Whole-cell response of the pennate diatom PhaeodactylUlII tri sequences in a conserved region of the D 1 protein satisfy comuturn to iron starvation. Proc. Nail. A cad. Sci. U. S. A. photosystem n function.]. Mol. Bioi. 246:120-31. 105: 10438-43. Kotakis, C., Petropoulou, Y., Stamatakis, K, Yiotis, C. & Manetas, Y. AlIen, A. E., Vardi, A. & Bowler, C. 2006. An ecological and 2006. 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Vass, I., Styring, S., Hundal, T., Koivuniemi, A., Aro, E.-M. & Lavaud, J 2007. Fast regulation of photosynthesis in diatoms: Andersson, B. 1992. Reversible and irreversible intermediates mechanisms, evolution and ecophysiology (a review). Funct. during photoinhibition of photosystem II: stable reduced ~ Plant Sci. Bioteel!. 1:267--87. species promote chlorophyll triplet formation. Proc. N(t/l. Acari. Lavaud,J & Kroth, P. 2006. In diatoms, the transthylakoid proton Sci. U. S. A. 89:1408-12. gradient regulates the photoprotective non-photochemical Vermaas, W., Vass, I., Eggers, B. & Styring, S. 1994. Mutation of a fluorescence quenching beyond its control on the xanthophyll putative ligand to the non-heme iron in photosystem II: cycle. Plant Cell Physiol. 47:1010-6. implications for Q(A) reactivity, electron transfer, and herbi Lavaud, J, Rousseau, B. & Etienne, A.-L. 2003. Enrichment of the cide binding. Biochim. Biophys. Acta 1184:263-72. light-harvesting complex in diadinoxanthin and implications Wagner, H., .Takob, T. & Wilhelm, C. 2006. Balancing the energy for the non photochemical quenching fluorescence quenching flow from captured light to biomass under fluctuating light in diatoms. Biochemistry U. S. A. 42:5802-8. conditions. New Phytol. 169:95-108. Lavaud, J, Rousseau, B., van Gorkom, H. & Etienne, A.-L. 2002a. Wilhelm, C., Biichel, C., Fisahn,J, Goss, R,.Takob, T., LaRoche,j., Influence in the diadinoxanthin pool size on photoprotection Lavaud,j., et aJ. 2006. The regulation of carbon and nutrient in the marine planktonic diatom Phaeodactylum triwrrtutum. assimilation in diatoms is significantly different fi-om green Plant Physiol. 129: 1398-406. algae. Protist 157:91-124. Lavaud,J, van Gorkom, H. & Etienne, A.-L. 2002b. Photosystem II Xiong, .T., Hutchison, R S., Sayre, R. T. & Govindjee. 1997. Modi electron transfer cycle and chlororespiration in planktonic fication of the photosystem II acceptor side function in a D 1 diatoms. Plwtosynth. Res. 74:51-9. mutant (arginine-269-glycine) of Chlamydomonas reinhanitii. Lazar, D. 2006. The polyphasic chlorophyll a fluorescence rise Biochim. Biophys. Acta 1322:60-76. measured under high intensity of exciting light. Funct. Plant Bioi. 33:9-30. Long, S., Humphries, S. & Falkowski, P. 1994. Photoinhibition of Supplementary Material photosynthesis in nature. Annu. Rev. Plant Physiol. Plant Mol. Bioi. 45:633-62. The following supplementary material is Maclntyre, H. L., Kana, T. M. & Geider, R. J 2000. The effect of available for this article: water motion on short-term rates of photosynthesis by marine phytoplankton. Trends Plant Sci. 5:12-7. Figure SI. Construction of the pGEM-T Dl Mann, D. G. 1999. The species concept in diatoms. Phycologia 38:437-95. transformation vectors. Mohanty, P., Allakhverdiev, S. I. & Murata, N. 2007. Application of low temperatures during photoinhibition allows characteriza Figure S2. Multiple sequence alignments and tion of individual steps in photodamage and the repair of phylogenetic reconstruction. photosystem n. Photosynth. Res. 94:217-24. Nishiyama, Y., Allakhverdiev, S. I. & Murata, N. 2006. A new paradigm Figure S3. Pair-wise comparison chart of Dl for the action of reactive oxygen species in the photoinhibition (PsbA) amino acid similarities and distances. of photosystem n. Biochim. Biophys. Acta 1757:742-9. Oettmeier, W. 1999. Herbicide resistance and supersensitivity in Figure S4. Pigment composition (in mol, photosystem n. Cell. Mol. Life Sci. 55:1255-77. 1 Ohad, N. & Hirshberg,.T. 1992. Mutations in the Dl subunit of 100 mol- chI a) and photosynthetic properties photosystem II distinguish between quinone and herbicide of the wildtype (WT) and the psbA mutants of binding sites. Plant Cell 4:273--82. Phaeodactylum tricornutum. Papageorgiou, G. c., Tsimilli-Michael, M. & Stamatakis, K. 2007. The fast and slow kinetics of chlorophyll a fluorescence Figure S5. Growth curves of the wild type (WT) induction in plants, algae and cyanobacteria: a viewpoint. and the four psbA mutants (V2l9I1F255I1S264A1 Photosynth. Res. 94:275-90. Paresys, G., Rigart, C., Rousseau, B., Wong, A. W. M., Fan, F., L275W) of Phaeodactylum tricornutum cells. Barbier, J-P. & Lavaud,.J. 2005. Quantitative and qualitative evaluation of phytoplankton populations by trichromatic chlorophyll fluorescence excitation with special focus on cyanobacteria. Water Res. 39:911-21. Perewoska, I., Etienne, A.-L., Miranda, T. & Kirilovsky, D. 1994. SI destabilization and higher sensitivity to light in metribuzin resistant mutants. Plant Physiol. 104:235--45. van Rensen,J.J. S., Xu, C. & Govindjee 1999. Role of bicarbonate in photosystem II, the water-plastoquinone oxido-reductase of plant photosynthesis. Physiol. Plant. 105:585-92. Sarthou, G., Timmermans, K. R, Blain, S. & Treguer, P. 2005. Growth physiology and fate of diatoms in the ocean: a review. J Sea Res. 53:25--41. FIG. SI. Construction of the pGEM-T Dl transformation vectors. AC codon264 Cc TpGEM-T DH3264T C . GG. A pGEM·T D1 ;.S264P Gc Gc A pGEM·T D1·S264G TXX A pGEM.TD1.S264A X TT TA A C A A C 6)~ BssSI pGEM~TD1 3795 bp 6 .A codon 268 G ~ T T· CG G Using the primers P-psbA196-5' (5'-CTGTTGCAGGTTCTTTATTATATGG-3') and P psbA990-3' (5'-TACTTCCATACCTAAATCAGCACGG-3'), a 795 bp fragment of the plastid encoded Phaeodactylum tricornutum gene psbA was amplified following standard PCR procedures (Sambrook et al. 1995) and subsequently ligated into the insertion site of the commercially available TA cloning vector pGEM-T according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA). The resulting vector pGEM-T Dl was subject to site-directed mutagenesis substituting the codon 264 (Ser) with four alternative codons that encode the amino acids Ala (pGEM-T Dl-S264A), Gly (pGEM-T Dl-S264G), Pro (pGEM-T Dl-S264P), and Thr (pGEM-T DI-S264T). Moreover, a BssSI recognition site was altered by a silent T to A nucleotide substitution in codon 268 (not altering the encoded amino acid sequence). Site-directed mutagenesis was performed via round circle PCR using the primers D 1- 792-fw (5' -CGTTTAA TCTTCCAA T ACGCTXXXTTTAACAACTCf!CGTGC-3 ') and DI-792-rev (5'- GCACGTGAGTTGTTAAAXXXAGCGTATTGGAAGATTAAACG- 3'), and the pGEM-T Dl vectors as template. The XXX within the primer sequence refers to the altered cod on (compared to the wildtype [WT] sequence) leading to the desired amino acid substitution. The small letter f! in the D 1-792-fw sequence indicates the second, conservative point mutation. Turbo-Pfu Polymerase (Stratagene, La Jolla, CA, USA) was used for the round circle PCR reaction. The resulting products were treated with DpnI for selective degradation of all nonmodified (hence methylated) parent strands and subsequently transformed into chemically competent Escherichia coli XL-I. The four resulting constructs served as transformation vectors in our attempts to transform the plastid genome of the diatom. FIG. S2. Multiple sequence alignments and phylogenetic reconstruction. A B The strong selection pressure acting on pshA led to a high degree of sequence conservation that allows reconstructing a phylogeny of photosynthetic organisms based on the PsbA (Dl) sequence that agrees well with more sophisticated phylogenetic reconstructions using more than one molecular marker (Delwiche 1999, Rodriguez- Ezpeleta et al. 2005). (A) Phylogenetic tree (neighbor joining, lOO-fold bootstrapping) of D 1 (PsbA) reconstructed from the amino acid sequence alignment [MUSCLE algorithm (Edgar 2004)] of35 individual protein sequences (see below for complete list). Although reflecting the current knowledge on plastid evolution in photosynthetic eukaryotes, the purpose of this phylogenetic reconstruction is not to draw evolutionary conclusions (few branches have bootstrap support of < 50), but rather it highlights the conservation level of D 1 and lists the species included in our analysis ofthe sequence conservation of the QB binding pocket. Three groups consisting of green algae, red algae, and glaucophytes directly derive from the primary endocytobiosis between a cyanobacterium and a eukaryotic heterotrophic cell. Euglenozoans and chlorarachniophytes are derived from secondary endocytobiosis between a green alga and a eukaryotic host cell. Secondary endocytobiotic engulfment of a red alga by a eukaryotic host gave rise to the heterokonts (or stramenopiles), cryptophytes, haptophytes, and dinoflagellates (Delwiche 1999, Cavalier-Smith 2000). (B) Sequence alignment of the QB pocket within the D1 protein. Light grey boxes: D, DE, and E helices; dark grey bars: His215 and His272, which bind a nonheme iron atom; red squares: localization of the point mutations (from left to right) V2191 / F2551 / S264A / L275W in Phaeodactylum tricornutum pshA mutants; light green boxes: amino acid substitutions to be transferred by the respective transformation vector. The conservation plot below the alignment shows the degree of conservation (in %) for every individual site. The blot was inferred from an amino acid sequence alignment ofD1 proteins from the 35 species listed in the phylogenetic tree above (Fig. S2A, or see complete list below). Synechocystis, Synechocystis sp. PCC6803 (cyanobacteria, NC_ 000911); Synechococcus WH7803 (cyanobacteria, CT971583); C. reinhardtii, Chlamydomonas reinhardtii (Chlorophytes, AF396929); A. thaliana, Arabidospsis thaliana (Embryophytes, CABI0554), C. merolae, Cyanidiosehyzon merolae 10D (Rhodophytes, BAC76132); T. pseudonana, Thalassiosirapseudonana (Heterokontophytes, Bacillariophyceae, EF067921); O. sinensis, Odontella sinensis (Heterokontophytes, Bacillariophyceae, CAA91657); P. tricornutum, Phaeodaetylum trieornutum (Heterokontophytes, Bacillariophyceae, AY864816). Materials and methods. All bioinformatic operations were performed using tools provided by the bioinformatic software package CLC Combined Workbench, Version 3.6.1 (CLC bio, Aarhus, Denmark). The phylogenetic tree for the Dl (PsbA) protein was reconstructed from an amino acid sequence alignment of entire D 1 protein sequences from 35 different species (Fig. S3). For the alignment of protein sequences the MUSCLE algorithm was used (standard settings, maximal 16 iterations). The neighbor-joining tree was reconstructed from the resulting alignment (data not shown); the value for the number of boots trap replicates was set to 100. The tree was rooted above the common ancestor of the cyanobacteria. From the same alignment a conservation blot was inferred. Therefore the graphical alignment settings of the CLC Combined Workbench were set to 'Line plot'. For Figure S2B, amino acid sequences of the quinone B (QB) pocket (amino acid position 189 to 298) from eight species (Fig. S3) plus the translated sequencing results from the four mutant strains were aligned using the MUSCLE algorithm (standard settings, maximal 16 iterations). Cavalier-Smith, T. 2000. Membrane heredity and early chloroplast evolution. Trends Plant Sei. 5: 174-82. Delwiche, C. F. 1999. Tracing the thread of plastid diversity through the tapestry of life. Am. Nat. 154:S164-77. Rodriguez-Ezpeleta, N., Brinkmann, H., Burey, S. C., Roure, B., Burger, G., Loffelhardt, W., Bohnert, H. J., Philippe, H., Lang, F. B. 2005. Monophyly of primary photosynthetic eukaryotes: Green plants, red algae and glaucophytes. Curr. BioI. 15: 1325-30. NCB! accession numbers ofPsbA (DJ) sequences (no particular order). All listed psbA genes were used for the MUSCLE amino acid alignment of the entire protein sequence, from which the phylogenetic tree (Fig. S2A) was reconstructed. The conservation plot (Fig. S4) has been derived from the same alignment. A subset (*) of the psbA genes listed was used for the MUSCLE amino acid alignment of the QB pocket (Fig. S2B). From another subset (t) the pair-wise comparison of amino acid similarities and distances was inferred (Fig. S4). ACCESSION CP000576 ORGANISM Prochlorococcus marinus str. MIT 9301 Bacteria; Cyanobacteria; Prochlorales; Prochlorococcaceae; Prochlorococcus. ACCESSION NC 000911 ORGANISM Syn~chocystis sp. pee 6803*t Bacteria; Cyanobacteria; Chroococcales; Synechocystis. ACCESSION CT971583 ORGANISM Synechococcus sp. WH 7803*t Bacteria; Cyanobacteria; Chroococcales; Synechococcus. ACCESSION BA000045 ORGANISM Gloeobacter violaceus pee 7421 Bacteria; Cyanobacteria; Gloeobacteria; Gloeobacterales; Gloeobacter. ACCESSION Z00044 S54304 ORGANISM Nicotiana tabacum Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; asterids; lamiids; Solanales; Solanaceae; Nicotianoideae; Nicotianeae; Nicotiana. ACCESSION X86563 ORGANISM Zea mays Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; ACCESSION EF044213 ORGANISM Coffea arabica Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; ACCESSION A Y228468 ORGANISM Pin us koraiensis Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Coniferopsida; Coniferales; Pinaceae; Pinus; Strobus. ACCESSION AY522329 ORGANISM Oryza sativa (indica cultivar-group) Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP ACCESSION AP000423 ORGANISM Arabidopsis thaliana*t Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; rosids; eurosids II; Brassicales; Brassicaceae; Arabidopsis. ACCESSION AB001684 ORGANISM Chlorella vulgaris t Eukaryota; Viridiplantae; Chlorophyta; Trebouxiophyceae; Chlorellales; Chlorellaceae; Chlorella. ACCESSION AF137379 L77928 ORGANISM Nephroselmis olivacea Eukaryota; Viridiplantae; Chlorophyta; Prasinophyceae; Pseudoscourfieldiales; Pycnococcaceae; Nephroselmis. ACCESSION DQ291132 ORGANISM Oltmannsiellopsis viridis Eukaryota; Viridiplantae; Chlorophyta; Oltmannsiellopsis. ACCESSION AP006715 ORGANISM Porphyra yezoensis Eukaryota; Rhodophyta; Bangiophyceae; Bangiales; Bangiaceae; Porphyra. . ACCESSION U38804 ORGANISM Porphyra purpurea t Eukaryota; Rhodophyta; Bangiophyceae; Bangiales; Bangiaceae; ACCESSION A Y673996 ORGANISM Gracilaria tenuistipitata var. liuit Eukaryota; Rhodophyta; Florideophyceae; Gracilariales; Gracilariaceae; Gracilaria. ACCESSION AF022186 Z36235 Z70297 ORGANISM Cyanidium caldarium Eukaryota; Rhodophyta; Bangiophyceae; Cyanidiales; Cyanidiaceae; Cyanidium. ACCESSION AB002583 ORGANISM Cyanidioschyzon merolae strain 10D*t Eukaryota; Rhodophyta; Bangiophyceae; Cyanidiales; Cyanidiaceae; Cyanidioschyzon. ACCESSION EF067921 ORGANISM Thalassiosira pseudonana*t Eukaryota; stramenopiles; Bacillariophyta; Coscinodiscophyceae; Thalassiosirophycidae; Thalassiosirales; Thalassiosiraceae; Thalassiosira. ACCESSION Z67753 ORGANISM Odontella sinensi*t Eukaryota; stramenopiles; Bacillariophyta; Coscinodiscophyceae; Biddulphiophycidae; Eupodiscales; Eupodiscaceae; Odontella. ACCESSION U30821 ORGANISM Cyanophora paradoxat Eukaryota; Glaucocystophyceae; Cyanophoraceae; Cyanophora. ACCESSION X70810 ORGANISM Euglena gracilis t Eukaryota; Euglenozoa; Euglenida; Euglenales; Euglena. ACCESSION AY741371 ORGANISM Emiliania huxleyit Eukaryota; Haptophyceae; Isochrysidales; Noelaerhabdaceae; Emiliania. ACCESSION DQ630521 L49157 ORGANISM Stigeoclonium helveticum Eukaryota; Viridiplantae; Chlorophyta; Chlorophyceae; ACCESSION DQ396875 L43360 ORGANISM Scenedesmus obliquus Eukaryota; Viridiplantae; Chlorophyta; Chlorophyceae; Sphaeropleales; Scenedesmaceae; Scenedesmus. ACCESSION AY835431 L44125 ORGANISM Pseudendoclonium akinetum Eukaryota; Viridiplantae; Chlorophyta; Ulvophyceae; Ulvales; Pseudendoclonium. ACCESSION NC 008289 ORGANISM Ost-;:eococcus taurit Eukaryota; Viridiplantae; Chlorophyta; Prasinophyceae; Mamiellales; Mamiellaceae; Ostreococcus. ACCESSION BK000554 AF396929 ORGANISM Chlamydomonas reinhardtii* Eukaryota; Viridiplantae; Chlorophyta; Chlorophyceae; Chlamydomonadales; Chlamydomonadaceae; Chlamydomonas. ACCESSION NC 005087 ORGANISM Physcomitrella patens subsp. patens Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Bryophyta; Moss Superclass V; Bryopsida; Funariidae; Funariales; Funariaceae; Physcomitrella. ACCESSION A Y864816 ORGANISM Phaeodactylum tricornutum*t Eukaryota; stramenopiles; Bacillariophyta; Bacillariophyceae; Bacillariophycidae; Naviculales; Phaeodactylaceae; Phaeodactylum. ACCESSION DQ851108 ORGANISM BigelowieUa natanst Eukaryota; Cercozoa; Chlorarachniophyceae; Bigelowiella. ACCESSION EF508371 ORGANISM Rhodomonas salina Eukaryota; Cryptophyta; Cryptomonadaceae; Rhodomonas. ACCESSION AF041468 AF063017 M76547 U81044 X14171 X14504 X51511 X52158 X52912 X56806 X62348 X62349 Z21976 ORGANISM Guillardia theta t Eukaryota; Cryptophyta; Ctyptomonadaceae; Guillardia. ACCESSION X56695 ORGANISM Ectocarpus siliculosus Eukaryota; stramenopiles; Phaeophyceae; Ectocarpales; Ectocarpaceae; Ectocarpus. ACCESSION AB027234 REGION: 4 .. 1086 ORGANISM Gymnodinium mikimotoit Eukaryota; Alveolata; Dinophyceae; Gymnodiniales; Gymnodiniaceae; Gymnodinium. FIG. S3. Pair-wise comparison chart ofDl (PsbA) amino acid similarities and distances. From a MUSCLE amino acid sequence alignment ofa set ofPsbA proteins (also see 83) a pair-wise comparison of amino acid similarities and distances was inferred. The similarities are shown in % within and above the fields forming the diagonal from top left to bottom right of the chart. The values below the diagonal represent the Jukes-Cantor distances (corrected for multiple substitutions) between the two compared sequences (Jukes and Cantor 1969). Red background indicates the highest values; blue background indicates the lowest values of the comparison. Highlighted by a black frame are the D 1 similarities of all listed species with the three diatoms. Materials and methods. All bioinformatic operations were performed using tools provided by the bioinformatic software package CLC Combined Workbench, Version 3.6.1 (CLC bio, Aarhus, Denmark). The pair-wise comparison of amino acid sequence similarities/distances for the QB pocket was inferred from a MUSCLE alignment (standard settings, maximal 16 iterations, alignment not shown) of the QB pocket sequences (amino acid position 198 to 307) from 18 different species (see Fig. S2). Jukes, T.& Cantor, C. 1969. Evolution of protein molecules. In Jukes, T. & Cantor, C. [Eds.] Mammalian Protein Metabolism. Academic Press, New York, pp. 21-32. FIG. S4. Pigment composition (in moIIlOO mol chI a) and photosynthetic properties of the wildtype (WT) and the pshA mutants of Phaeodactylum tricornutum. Pigment/Parameter WT V2191 F2551 S264A L275W DCMU resistance / 3 150 3000 500 ChI a (pg cell-I) 0.50 ± 0.07 0.57 ± 0.08 0.53 ± 0.17 0.54 ± 0.03 0.57 ± 0.1 Diadinoxanthin 9.4±2.1 8.2 ± 2.5 8.3 ± 0.4 8.4 ± 1.3 9.0 ± 2.1 Fucoxanthin 71.3 ± 5.7 74.3 ± 4.9 75.5 ± 2.8 69.4 ± 3.4 76.6 ± 4.0 ChI c 13.7 ± 0.7 14.7 ± 0.4 14.9 ± 1.3 14.2 ± 0.7 15.9 ± 1.7 l3-carotene 7.7 ±0.7 6.7 ± 1.0 6.6 ± 0.4 6.5 ± 1.3 6.0 ± 0.1 Yss (/lg ChI a-I) 90 ± 11 103 ± 10 136±7 149 ± 11 47±4 RC/CSo 158 ± 4 194±4 216 ± 2 232 ± 1 n.d. PSI:PSII 0.44 ± 0.04 0.46 ± 0.02 0.47 ± 0.03 0.43 ± 0.03 0.56 ± 0.07 111112 of Y ss 0.58 0.58 0.45 0.35 0.40 2 I EK (/lmoI photons'm- 's- ) 183 ± 5 ' 189 ± 10 223 ± 20 240 ± 10 231 ± 10 J (QAQB-/QA-QB-) 0.56 ± 0.06 0.61 ± 0.07 0.69 ± 0.09 0.96 ± 0.03* 0.88 ± 0.09 2 I (QA-QB ") 0.89 ± 0.02 0.82 ± 0.03 0.82 ± 0.04 0.95 ± 0.02 0.81 ± 0.03 Misses/PSII 0.14 ± 0.01 0.14±0.01 0.17 ± 0.01 0.23 ± 0.01 n.d. Fo 131±6 141 ± 11 139 ± 7.5 170 ± 9 180 ± 7 Fv/Fm 0.70 ± 0.02 0.68 ± 0.01 0.69 ± 0.01 0.71 ± 0.03 0.57 ± 0.01 ETo/CSo 182 ± 17 167 ± 4 145 ± 25 89 ± 17 75 ±28 I /l (d- ) 2.54 ± 0.2 2.72 ± 0.2 2.44 ± 0.3 2.38 ± 0.2 1.87 ± 0.1 Yss is the concentration of active PSII reaction centers per chI a (Lavaud et al. 2002a); RC/CSo is the number of active PSII reaction center per PSII cross-section; 1IIl/2 of Y ss is a measurement of the PSII light-harvesting antenna size (Lavaud et al. 2002a); EK is the light intensity for saturation of photosynthesis; the DCMU resistance factor was calculated from the IC50 PSII fluorescence reactivity to DCMU; J (* J phase is delayed to 9.7 ± 2.5 ms in S264A) and I are the fluorescence levels ofthe J and I phases measured at 2 ms and 30 ms, respectively (values normalized to the P peak = 1, see Fig. 4A, P corresponds to the 2 concentration ofQA-QB - and PQH2); Fo is the minimal level of fluorescence of dark-adapted cells; Fv/Fm is the maximum photosynthetic efficiency ofPSII; PSII [(Fm'-P) I Fm'] is the effective PSII quantum yield for photochemistry measured at 50-100 !-lmol photons'm-2's- 1 (an irradiance for which there is no NPQ); ETolCSo is the steady-state electron transport in a PSII cross-section; !-l is the growth rate. All measurements were performed on cells grown at 50 2 l !-lmol photons'm- 's- , which was low enough to prevent the de-epoxidation of diadinoxanthin into diatoxanthin. n. d., not determined. Values are average ± SD of three to four measurements. See the text for other details. Materials and methods. DCMU resistance was evaluated measuring the inhibition of the PSII activity versus increasing DCMU concentrations using a self-made fluorometer (Paresys et al. 2005). The measure of inhibition is the increase in the amplitude of the 1-45 ms peak illustrating the accumulation ofQA- due to the electron QA-QB transfer being blocked by DCMU (see Fig. 4B). This way, the IC5o, the DCMU concentration at which the PSII activity is inhibited by 50% was measured and compared between WT and mutants to evaluate a resistance factor. Standard fluorescence nomenclature was used. Fo and Fm are defined as the minimum PSII fluorescence yield of dark-adapted cells and the maximum PSII fluorescence yield reached in such cells during a saturating pvlse of white light, respectively. The PSII quantum yield for photochemistry is the ratio Fv/Fm where Fy is the variable part ofthe fluorescence emission and is equal to Fm-Fo. The photochemical quenching was qP = (Fm-P)/(Fm'-Fo'), where P is the steady-state level of fluorescence emission reached after few minutes of illumination. l--qP is a parameter to estimate the fraction of reduced QA (BUchel and Wilhelm 1993, Niyogi et al. 1998). A classic OIJP analysis was also performed (Strasser et al. 1995, Force et al. 2003) allowing to extract the following data: time to reach the J phase (QAQB-/QA-QB- state), time to 2 reach the 1 phase (QA-QB - state), and the parameters RC/CSo and ETo/CSo (Force et al. 2003). For the control measurement of the relative O2 yield produced per flash during a sequence of single-turnover saturating flashes (02 sequences), cells were first dark-adapted for 20 min and then deposited on the electrode. The cells were allowed to settle on the electrode for 7 min in darkness before measurement. The steady-state O2 yield per flash (Y ss) was attained for the last four flashes of a sequence of 20 flashes when the classical four-step oscillations due to the S-state cycle ofthe PSII RC 02-evolving complex (Kok et al. 1970; see Fig. 4 for a classic recording) were fully damped. The oscillation is damped after a few cycles to a constant steady-state yield, Y ss, due to loss of synchrony caused mainly by a nonzero probability of "misses" (reflecting the number ofPSlI RCs unable to perform a charge separation during a flash, Lavorel 1976). Yss was used to evaluate the number of O2- producing PSII RCs relative to chI a (see Lavaud et al. 2002). The miss probability per PSlI was calculated from the Lavorel matrix (Lavorel 1976). For the saturation curves of Y ss, the intensity of the flashes was varied with neutral density filters. A new dark-adapted sample was used for each flash intensity measurement. 111112 of Y ss, the reciprocal of the half-saturating flash intensity of flash O2 evolution saturation curves was used as a measure of the PSII antenna size (Lavaud et al. 2002). EK, the irradiance for saturation of photosynthetic 02 emission was estimated from PIE curves. BUchel, C. & Wilhelm, C. 1993. In vivo analysis of slow chlorophyll fluorescence induction kinetics in algae: progress, problems and perspectives. Photochem. Photobiol. 58: 137-48. Force, L., Critchley,C. & van Rensen, J. J. S. 2003. New fluorescence parameters for monitoring photosynthesis in plants. 1. The effect of illumination on the fluorescence parameters of the JIP-test. Photosynth. Res. 78: 17-33. Kok, B., Forbush, B. & McGloin, M. 1970. Cooperation of charges in photosynthetic O2 evolution-I. A linear four step mechanism. Photochem. Photobiol. 11 :457-75. Lavaud, J., van Gorkom, H. & Etienne, A.-L. 2002. Phbtosystem Il electron transfer cycle and chlororespiration in planktonic diatoms. Photosynth. Res. 74:51-9. Lavorel, J. 1976. Matrix analysis of the oxygen evolving system of photosynthesis. J. Theor. Bioi. 57:171-85. Niyogi, K. K., Grossman, A. R. & Bjorkman, O. 1998. Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell 10:1121-34. Paresys, G., Rigart, C., Rousseau, B., Wong, A. W. M., Fan, F., Barbier, J.-P. & Lavaud, J. 2005. Quantitative and qualitative evaluation ofphytoplankton populations by trichromatic chlorophyll fluorescence excitation with special focus on cyanobacteria. Water Res. 39:911- 21. Strasser, R. J., Srivasatava, A. & Govindjee. 1995. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 61 :32-4.