chlorophyll-protein complexes from the red-tide ... · chlorophyll a, chlorophyll c2, and peridinin...

Plant Physiol. (1987) 83, 805-8120032-0889/87/83/0805/08/$0 1.00/0

Chlorophyll-Protein Complexes from the Red-TideDinoflagellate, Gonyaulax polyedra Stein'ISOLATION, CHARACTERIZATION, AND THE EFFECT OF GROWTH IRRADIANCE ON CHLOROPHYLLDISTRIBUTION

Received for publication July 28, 1986 and in revised form November 5, 1986

BARBARA A. BOCZAR*2 AND BARBARA B. PREZELINOceanic Biology Group, Marine Science Institute and Department ofBiological Sciences, University ofCalifornia, Santa Barbara, California 93106

ABSTRACTI

A comparision of high (330 microeinsteins per meter squared persecond) and low (80 microeinsteins per meter squared per second) lightgrown Gonyaulax polyedra indicated a change in the distribution ofchlorophyll a, chlorophyll c2, and peridinin among detergent-solublechlorophyll-protein complexes. Thylakoid fractions were prepared bysonication and centrifugation. Chlorophyll-protein complexes were solu-bilized from the membranes with sodium dodecyl sulfate and resolved byDeriphat electrophoresis. Low light cells yielded five distinct chlorophyll-protein complexes (I to V), while only four (I' to IV') were evident inpreparations of high light cells. Both high molecular weight complexes Iand I' were dominated by chlorophyll a absorption and associated withminor amounts of chlorophyll c. Both complexes II and II' were chloro-phyll a-chlorophyll c2-protein complexes devoid of peridinin and uniqueto dinoflagellates. The chlorophyll axc2 molar ratio of both complexeswas 1:3, indicating significant chlorophyll c enrichment over thylakoidmembrane chlorophyll a.c ratios of 1.8 to 2:1. Low light complex IIIdiffered from all other high or low light complexes in that it possessedperidinin and had a chlorophyll a.c2 ratio of 1:1. Low light complexes IVand V and high light complexes III' and IV' were spectrally similar, hadhigh chlorophyll a.c2 ratios (4:1), and were associated with peridinin. Theeffects of growth irradiance on the composition of chlorophyll-proteincomplexes in Gonyaulax polyedra differed from those described for otherchlorophyll c-containing plant species.

An increasing number of investigators have become interestedin the isolation ofpigment protein complexes from the thylakoidmembranes of the chromophytic algae. Chromophytic algaetypically contain Chl c as their accessory Chl and may haveaccessory carotenoids, such as peridinin or fucoxanthin, thatefficiently transfer absorbed light energy to photosynthetic reac-tion centers (10, 27). Particular attention has been given to Chla-Chl c1-Chl c2-fucoxanthin containing diatoms and brownmacroalgae, where the P700-Chl a-protein complex of PSI reac-tion centers has been isolated (1, 18). Barrett and Anderson (3,4) have also characterized two light-harvesting pigment-protein

'Supported by United States Department of Agriculture PL95-224and by National Sciences Foundation OCE-82-08187 and OCE-84-08933.

2Present address: Department of Botany KB- 15, University of Wash-ington, Seattle, WA 98195.

complexes (LHC3) from brown macroalgae, including a Chl a-Chl c-fucoxanthin complex and a Chl a-Chl c complex. Recently,Friedman and Alberte (8) and Owens and Wold (18) haveisolated a LHC from the diatom, Phaeodactylum tricornutumwhich contained Chl a, Chl c, and fucoxanthin. Owens and Wold(18) also demonstrated the presence of a distinct Chl a-Chl cLHC in P. tricornutum which was devoid of fucoxanthin.

Photosynthetic dinoflagellates, a major group of primary pro-ducers in the marine environment, also possess the Chl a-Chl c-carotenoid pigment system characteristic of many groups ofaquatic primary producers. Dinoflagellates, however, possess thecarotenoid peridinin instead of fucoxanthin and have only onetype of Chl c, i.e. Chl c2 (12). These light-harvesting pigmentsare associated with the various Chl a-protein complexes withinthe thylakoid membranes. Complexes containing the reaction*center of PSI have been isolated from two species of dinoflagel-lates (22). These P700-Chl a-protein complexes have spectraland kinetic properties similar to PSI complexes isolated fromother plant species. In addition, a major light-harvesting complexof dinoflagellates has been characterized as a water-soluble peri-dinin-Chl a-protein complex (PCP). The PCP complex containsthe majority of cellular peridinin, up to 20% of the cellular Chla and is devoid of all other photosynthetic pigments found inthe chloroplast (cf 21). The species distribution, photophysiol-ogy, and molecular topology of PCP has been extensively char-acterized (19-26). Recently, a second major light-harvestingcomplex was isolated from three dinoflagellate species and char-acterized as a Chl a-Chl c2-protein complex devoid of peridininbut which transferred absorbed light energy from Chl c2 to Chla (5-7). The organization of Chl-protein complexes within thethylakoid membranes of dinoflagellates has been modelled afterthat described for vascular plants (22, 28, 29).The photosynthetic adaptation of unicellular algae to various

light intensities has been documented in a wide variety of algalgroups (cf 23). Cellular responses to a change in light intensitycan include changes in pigment concentration and/or composi-tion, photosynthetic unit density and optical cross-section, andphotosynthetic carbon fixation parameters (cf 20). Recent stud-ies of the diatom, P. tricornutum documented changes in themajor detergent-soluble light harvesting complex (18) and itsapoprotein (9) in response to changes in light intensity. Thepresent study characterizes the biochemical features of Chl a-protein complexes isolated from populations of the red-tidedinoflagellate, Gonyaulax polyedra grown under high and low

3 Abbreviations: LHC, light-harvesting complex; LL, low light; HL,high light; PCP, peridinin-chlorophyll a-protein; PMSF, phenylmethyl-sulfonylfluoride.

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BOCZAR AND PREZELIN

irradiances. Results indicate that growth irradiance does influ-ence the distribution of pigments in the Chl a-protein compexesof G. polyedra.

MATERIALS AND METHODS

Growth Conditions. Low light (LL, 80 uE m-2 s-') and highlight (HL, 330 ME m-2 s-') cultures of Gonyaulaxpolyedra Stein,clone 70A (University of California, Santa Barbara, UCSB CodeNo. 5M20) were grown in unialgal batch cultures in 2.8 LFembach flasks containing 1.5 L of f/2 medium (15). Cells werecultured in an alternating 12 h light, 12 h dark schedule at 22°C± 1°C. Illumination was provided by General Electric cool-whitefluorescence lamps. Doubling times were 4 to 5 d for bothpopulations, which were harvested in mid-log phase (4-5 x 103cells/ml) by centrifugation (15C, 20 min, I000g). Pelleted cellswere resuspended in 50 mM Tris/HCl-l mM EDTA- I mM PMSF(pH.8.0).

Preparation and Solubilization of Thylakoid Membranes. Cellsuspensions were sonicated at 0°C (Sonifier model W185) until80 to 90% cell breakage was determined microscopically. Wholecells and large cellular debris were sedimented by centrifugationat 500g, 10 min, 4C. Thylakoid membranes were pelleted bycentrifugation (30,000g, 70 min, 4C) and the pellet washed byrecentrifugation following resuspension in 50 mM Tris/HCl-lmM EDTA-l mm PMSF (pH 8.0).Membranes were solubilized in 1.5% SDS at an SDS:Chl a

ratio of 22:1 and centrifuged at 38,000g for 15 min at 40C. SDS-

Table I. Electrophoretic and Pigmentation Properties ofChi a-ProteinComplexes Isolatedfrom LL (80 ME m2 s') Grown G. polyedra.

Cellular Chl a:c2 was 2:1

Band Position

I II III IV&V

Mobility 0.04 0.32 0.51 0.76 0.89Approximate molwt (kD) 133 92 71 51 46

% Chl aa 48 (3.0)b 7 (0.4) 12 (4) 34 (4.7)% Chl c2c 17 44 22 17Chl a:c2 (molar

ratio) 5.0 0.32 (0.1) 1.01 (0.5) 4.0 (0.5)a Percentage of total Chl a on the Deriphat gel. b Numbers in

parentheses indicate standard deviations from the mean; n = 5 in allcases. c Percentage of total Chl c was calculated from the Chl a:c2molar ratio of the SDS supernatant and the % Chl a and Chl a:c ratio ofthe complex.

Table II. Electrophoretic and Pigmentation Properties ofChl a-ProteinComplexes Isolatedfrom HL (330 ME m2 s') Grown G. polyedra.

Cellular Chl a:c2 Was 1.8:1

Band Position

I' II' III' IV'Mobility 0.09 0.30 0.45 0.76Approximate molwt(kD) 125 95 78 51

% Chl aa 37 (1.5)b 14 (0.8) 17 (1.1) 32 (0.5)%Chlc2c 12 66 10 12Chl a:c2 (molar

ratio) 4.5 (1.5) 0.33 (.1) 2.5 (.5) 4.0 (.5)aPercentage of total Chl a on the Deriphat gel. b Numbers in

parentheses indicate standard deviations from the mean; n = 5 in allcases. c Percentage of totalkChl c was calculated from the Chl a:c2molar ratio of the SDS supernatant and the % Chl a and Chl a:c ratioof the complex.

solubilized membrane extracts were recovered from the super-natant and loaded onto either polyacrylamide tube gels (0.5 x8.0 cm) or a slab gel (2.5 x 115 mm). Both electrophoretic gelsystems contained 5% acrylamide (Bio-Rad), 0.25% N,N-meth-ylene bis acrylamide (Bio-Rad), 0.125% ammonium persulfate(Sigma), 0.1% (v/v) N,N,N',N'-tetra methylethylene diamine(Eastman Kodak), 6.2 mM Tris, 48 mM glycine, and 0.1% Deri-phat 160C (partial sodium salt of N-lauryl B-iminodipropionicacid, Gift from the Henckel Corporation). The quality of Deri-phat 160C varied from lot to lot, with two of five lots testedfound to be unsuitable for experimental work. Electrophoresiswas carried out at 75 V for 35 to 45 min at room temperature,using an electrode buffer containing 12.4 mm Tris, 96 mMglycine, and 0.2% Deriphat 160C.

Following electrophoresis, protein bands were detected byfixing Deriphat tube gels overnight in 10% acetic acid, proteinstaining with 0.2% Coomassie blue R in methanol:H20:aceticacid (5:5:1 v/v/v) and destaining the gel background with meth-anol:H20:acetic acid (5:5:1 v/v/v). Relative Chl a distribution inthe Deriphat gel was determined from gel scans measured at 670nm with a Beckman Acta III spectrophotometer. Gel scan pat-terns were photocopied and individual peaks were cut andweighed. Chl a-containing bands were then excised from tubegels and either used immediately, stored at -76°C for futureprotein analyses, or the Chl a-containing components were elutedfrom the gel with 0.3% SDS in 50 mm Tris (pH 8.0) by methodspreviously described (6). Deriphat-containing slab gels were usedto determine the relative mobility ofsolubilized Chl a-containingcomponents as compared to standard proteins. Marker proteins(Bio-Rad) included: myosin (200 kD); ,B-galactosidase (130 kD);phosphorylase b (92 kD); BSA (68 kD); ovalbumin (43 kD);carbonic anhydrase (31 kD); trypsin inhibitor (21 kD); andlysozyme (14 kD). All protein markers were denatured prior toelectrophoresis by boiling solutions for 3 min in 6.2 mm Tris, 48mm glycine, 1% SDS, 1% 2-mercaptoethanol, 10% (v/v) glycerol.Protein-stained slab gels, prepared in an identical manner as tubegels, were used to estimate mol wt of the native complexes.

Pigmentation. Chl a:c2 molar ratios were determined using theacetone extraction procedures detailed in Prezelin and Haxo(24). In addition, all resolvable complexes were eluted from the

EcC14N(-(D-

.0

0

(U

0 0.5 1.0

Mobility

FIG. 1. Deriphat polyacrylamide gel scan (672 nm) of Chl-a-proteincomplexes isolated from cultured populations of G. polyedra grown at(a) LL (80 uE m-2 s-') and (b) HL (330 ME m-2 s-') intensities.

806 -Plaant Physiol. Vol. 83, 1987

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CHLOROPHYLL-PROTEIN COMPLEXES FROM GONYAULAX POLYEDRA STEIN

Wavelength (nm)

as0C0

0.01

a1)

-ucc

400 500 600Wavelength (nm)

500 600 700 400 500 600Wavelength (nm) Wavelength (nm)

gel and pigments extracted with 90% (v/v) acetone. The clarifiedsupernatant was transferred to diethyl ether, and concentratedunder N2 for further chromatographic analyses ofcarotenoid andChl components. One-dimensional TLC was carried out oncellulose plates according to the methods of Jeffrey (13).

Protein Analysis. Gel slices of each Chl a-containing bandwere either used immediately for protein analyses or frozen forsubsequent use. In either case, gel slices were equilibrated for 15to 20 min in a solution of 50 mm Tris, 3% SDS, and 1% 2-mercaptoethanol. The gel slices were then heated for 2 min at70°C and reelectrophoresed, using a Laemmli buffer system witha 12% resolving gel and a 3% stacking gel (14). Protein bandswere resolved using the silver-staining technique described byMorrissey (17) and gels were scanned with an ISCO model 1310

700 FIG. 2. A comparison ofroom temperatureabsorption spectra for (a) LL (80 ME m2 s-)complex I, (b) HL (330 ME m-2 s-') complexI', (c) LL complex II, and (d) HL complex

1II' isolated from LL and HL grown popula-tions of G. polyedra.

gel scanner with an ISCO UA-5 absorbance/fluorescence moni-tor. Mol wt protein standards were the same as those used in theslab gel system.

Absorption and Fluorescence (Emission and Excitation) Spec-tra. Absorption spectra were recorded using an Aminco DW2spectrophotometer. Room temperature fluorescence emissionand excitation spectra of gel slices suspended in 50 mm Tris (pH8), or of solutions of eluted Chl a-protein complexes, wererecorded on a Perkin-Elmer MPF-2A spectrofluorimeter. Fluo-rescence data were relayed to a Bascom-Turner microprocessorat 2 nm intervals and subsequently displayed on the X-Y plotterof an Aminco DW2 spectrophotometer. Excitation spectra werequanta corrected for wavelength variation in xenon lamp energyoutput. Emission spectra were corrected for instrument sensitiv-

807

a)C.)Co

0

0

._

co

6

a)

ca

4)

cc

0C

.0100)

.0

cc

400



BOCZAR AND PREZELIN

_A600 650 700

Wavelength(nm)

FIG. 3. A comparison of room temperature fluorescence emissionspectra of LL (80 ME m-2 s-',-) complex I gel slices and HL (330 MEm-2 s-', ---) complex I' gel slices isolated from SDS/Deriphat prepa-rations of LL and HL grown populations of G. polyedra. Excitationwavelength was 435 nm.

ity. Fluorescence measurements were made on samples with Chla red absorption maxima less than 0.1 O.D. Excitation slit widthswere between 2 and 8 nm, and emission slit widths were between2 and 4 nm.

RESULTS

Low and high light grown Gonyaulax polyedra populationshad similar whole cell Chl a:c molar ratios, i.e. 2:1 and 1.8:1,respectively (Tables I and II). In LL cultures, the Chl content oflog phase cells was about twice as concentrated as those measuredfor cells growing under HL intensities. The average Chl a and ccontent of LL cells was about 40 ± 4 and 20 ± 2 gM cell-' (109)(n=3), respectively. Previous studies (26) also indicated that theperidinin content of LL G. polyedra is up to 2-fold greater thanthat ofHL cells. When thylakoid membranes isolated from eitherLL and HL cultures were solubilized with 1.5% SDS, more than85% ofthe total Chl a in the detergent homogenate was recoveredin the SDS supernatant. Analyzed just prior to Deriphat electro-phoresis, Chl a:c molar ratios of solubilized thylakoid membranefractions were 1.9:1 and 1.6:1, respectively, for LL and HLpreparations.

Five Chl a-protein complexes were resolved from LL G. pa.lyedra preparations (Fig. la), although the resolution ofcomplex

V varied. Approximately half of the gel Chl a was associatedwith complex I (48%) and an additional third (34%) with com-plexes IV and V (Table I). The remainder of the Chl a wasdivided between complexes 11 (7%) and III (12%). Chl c wasassociated with all complexes, but was concentrated in complexII (44%), while peridinin was associated with complexes II, III,and IV. By comparison, HL grown populations of G. polyedrahad four resolvable Chl a-protein complexes (Fig. lb). Like LLcells, the majority of total Chl a in HL cells was associated withcomplexes I' and IV' (Table II). However, there was a signifi-cantly greater gel Chl a (14%) and Chl c (66%) associated withcomplex II' in HL cells than observed in the comparable complexII of LL cells. By comparison, there was no detectable change inthe percent gel Chl a associated with HL complex III' over thatseen in LL complex III, but the percent gel Chl c in complex III'was only half that of complex III. All pigmented bands on theDeriphat gels contained protein components which stained withCoomassie brilliant blue R.

COMPARISON OF chl a-PROTEIN COMPLEXES

Complexes I and I'. LL complex I was green in color andcharacterized by a room temperature absorption spectrum dom-inated by Chl a absorption (438 nm, 672 nm) (Fig. 2a). Thepresence of some Chl c2, which contributed to the small 640 nmpeak as well as to blue light absorption around 450 nm, wasconfirmed by TLC. The Chl a:c molar ratio of complex I was5:1, representing a 2.5-fold enrichment over that of the initialmembrane preparation. Peridinin was not detectable, althoughTLC indicated the presence of yellow xanthophylls and f3-caro-tene which presumably accounted for the strong absorptionshoulder at 485 nm. The HL complex I' (Fig. 2b) was almostspectrally identical to complex I and TLC indicated the samepigment components were present in both complexes. The onlyapparent change was a 2 nm blue shift in the blue absorptionpeak of complex I'. But while spectrally similar, the percent gelChl a was lower in complex I' (37%) compared to complex I(48%). The Chl c content was equally lower in complex I', sothat the Chl a:c molar ratio of complex I' was comparable tothat of complex I.The Chl a room temperature fluorescence emission maximum

of complex I was centered at 675 nm (excitation at 435 nm),while the emission at 640 nm wLs indicative of energeticallyuncoupled Chl c (Fig. 3). By comparison, complex I' had a 675nm Chl a emission peak, but uncoupled Chl c fluorescence at640 nm was not evident.When LL complex I was re-electrophoresed on Laemmli gels,

FIG. 4. Laemmli gel silver stain patterns (scanned at546 nm) of polypeptides isolated from G. polyedra (a)LL (80 ME m-2 s-') complexes I and II, and (b) HL (330ME m-2 s-') complexes I' and II'. Determined mol wtshown for each polypeptide band is expressed as kD.

MOBILITY

808 Plant Physiol. Vol. 83, 1987

co

..r

MOBI LITY




E-2 L

~~~~~~~~~~~640

400 500 600 600 700Wavelenigth (n m}

FIG. 5. Room temperature (a) fluorescence excitation and (b) emis-sion spectra of complex II gel slices isolated from SDS/Deriphat prepa-rations of LL (80,uE m-2 s-') grown G. polyedra. Excitation wavelengthat 435 nm. Emission wavelength at 673 nm.

a major high mol wt polypeptide component (106 kD) wasresolved which could not be further dissociated by additionalheating or by increasing SDS or 2-mercaptoethanol concentra-tions (Fig. 4a). Such a high mol wt polypeptide was not evidentin similar preparations of complex I' (Fig. 4b). Two majorpolypeptides of 61 and 55 kD were also associated with complexI while the major polypeptide associated with high light complexI appeared to be 70 kD. Four additional complex Y polypeptideswere resolved at 74, 59, 23, and 16 kD, but not evident in similarpreparations of complex I.Complexes II and II. While Chl c2 appeared to be present in

all low light pigment-protein complexes isolated by SDS solubi-lization and Deriphat electrophoresis, the largest percentage(44%) of Chl c was concentrated in the apple-green coloredcomplex II (Tables). The absorption spectrum ofcomplex II wasdominated by a blue peak at 452 nm and absorption peaks at672 and 635 nm (Fig. 2c). The Chl aoy molar ratio was 0.32:1.TLC analyses verified the absence of peridinin and the presenceofyellow xanthophylls. Major polypeptide components occurredat 49 and 38 kD on Laemmli gels, with others detected at 32,21, and 16 kD (Fig. 4a). The excitation spectrum ofLL complexII indicated some coupling of light absorbed by Chl c (A 450-460) to Chl a fluorescence emission at 675 nm (Fig). However,the fluorescence emission spectrum indicated that some fractionof Chl c associated with complex II was energetically uncoupledfrom Chl a, as evidenced by the Chl c emission peak at 640 nm.The electrophoretic mobility, chromophore ratio, approximate

mol wt, and spectral characteristics of complex II were identicalin HL cells. However, the percentage of total gel Chl a and Chlc associated with HL complexI.' was 100 and 50%, respectively,greater than that ofcomplexWI. There were also major differencesin polypeptide band pattens ofcomplexes II and by' on Laemmligels. Five polypeptides with mol wt less than 50 kD were resolvedin preparations of complex (Fig. 4a). By comparison, threemajor HL complex IIIpolypeptides were resolved at 72, 60, and51 kD, with two additional minor polypeptides were detectableat 41 and 38 kD (Fig. 4b).Complexes III and the'. LL complex III was spectrally distinct

from any detergent-solubilized complex previously isolated froma dinoflagellate (5). It had an equal proportion of both Chls,giving complex III a Chl a:c molar ratio of 1: 1, and accountedfor 22% of the total gel Chl c found on a typical Deriphat gel(Tablef). The absorption spectrum was dominated by a majorpeak associated with Chl c, as well as a Chl a red peak at 672nm (Fig. 6a). In contrast to the higher mol wt complexes I andIn described above, TLC analyses indicated the presence of

peridinin in complex III. Peridinin presumably accounted forthe orange color of the complex, and the 485 nm shoulder in theabsorption spectrum. While HL complex III' had a mobility anda mol wt approximating that ofLL complex III, it had distinctlydifferent spectral characteristics. The Chl a red peak remainedat 672 nm but there were two peaks at 432 and 450 nm whichdominated the blue region of the absorption spectrum (Fig. 6b).TLC analyses confirmed the presence of Chl c in complex III',which had a high a:c molar ratio of 2.5:1. Peridinin was alsopresent, contributing to the orange color of the complex and theabsorption shoulder centered around 485 nm.The fluorescence excitation spectrum (Fig.7a) for the Chl a

emission peak of complex III (Fig. 7b) indicated that both Chl c(A 450-460) and peridinin (A 460-520) transferred absorbedlight energy to Chl a. The secondary 640 nm peak in thefluorescence emmision spectrum (Fig. 7b) indicated that a frac-tion of the total Chl c present was energetically uncoupled. OnLaemmli gels, a 45 kD polypeptide appeared to be a majorcomponent in the complex III, with associated polypeptides at33 and 24 kD (Fig. 8a). Complex III' polypeptides patterns alsoincluded 45 kD and 32 to 35 kD bands, as well as a distinct 57kD polypeptide not seen in LL complex III (Fig. 8b).Complexes IV, V, and IV'. Taken together, the brick-red LL

complexes IV and V contained about 17% of the gel Chl c andtheir combined Chl a:c molar ratio approached 4:1 (Table I).These spectrally similar complexes (Fig. 6, c and d) containedperidinin, accounting for their color and spectral absorptionbetween 480 and 520 nm. HL complex IV' had a mobility, anapproxiamte mol wt, and a Chl a:c molar ratio similar to that ofLL complex IV + V. While TLC analyses indicated that peridininwas present in complex IV', the markedly reduced spectralabsorption in the carotenoid region between 480 to 520 nmsuggested the relative amounts of peridinin were reduced in HLcomplex IV' over analogous LL complexes. LL complexes IVand V both contained polypeptide components in the 24 to 37kD range, as well as two lower mol wt polypeptides at 17 and 19kD (Fig. 8a). HL complex IV' had a polypeptide band patternsimilar to LL complex V, with four polypeptide bands beingresolved at 33, 23, 18, and 16 kD (Fig. 8b).

DISCUSSIONFew investigators have attempted to isolate and characterize

the membrane-associated Chl-protein complexes of dinoflagel-lates. Using the detergent Triton X- 100 and column chromatog-raphy, Prezelin and Alberte (22) were able to isolate P700-Chl a-protein complexes from two marine dinoflagellates, Glenodiniumsp. and Gonyaulax polyedra. However, they were unable tocharacterize any membrane-associated light-harvesting compo-nents by this method or by SDS electrophoresis. By using theSDS-solubilization/Deriphat electrophoresis method of Mark-well et al. (16), Boczar and coworkers (6, 7) were able to separatethe majority ofGlenodinium sp. thylakoid Chl a into four distinctChl a-protein complexes. Identical methods were used in thepresent study to separate Chl a-protein complexes from the redtide dinoflagellate, Gonyaulax polyedra. Results indicated thatboth the number and composition of Chl a-protein complexesin G. polyedra were influenced by growth irradiance, while thebiochemical and spectral characteristics of these Chl a-proteincomplexes differ in detail from those described for cells ofGlenodinum sp. cultured under similar light intensities (6).Low light grown G. polyedra cells contained five SDS/Deriphat

Chl a-protein complexes (I-V), while only four (I'-IV') wereevident in identical preparations of high light cells. Both highmol wt Deriphat complexes I and I' were spectrally similar, butnot identical, to the P700-Chl a complex routinely isolated fromthe PSI reaction center ofmany plant groups by SDS electropho-resis (28-30). But unlike the P700-Chl a-protein complex previ-

809



BOCZAR AND PREZELIN

Wavelength (nm)

Plant Physiol. Vol. 83, 1987

FIG. 6. A comparison of room temperature absorp-tion spectra for (a) LL (80 uE m-2 s9') complex III, (b)HL (330 uE m-2 s-') complex III', (c) LL complex IV,(d) LL complex V, and (e) HL complex IV' isolatedfrom LL and HL grown populations of G. polyedra.

Wavelength (nm)

0c.0.00)

cc-

I I


810

0C1)00

En

0D

z

c20n

D

._i0

cc

C)

._

co

0

.0

Cc


700

700




0 '0520 673

*~~~~~~~~~~~~640

400 500 600 600 700Wavelength min)

FIG. 7. Room temperature (a) fluorescence excitation and (b) emis-sion spectra of complex III gel slices isolated from SDS/Deriphat prepa-rations of LL (80 ME m-2 s-') grown G. polyedra. Excitation wavelengthat 435 nm. Emission wavelength at 673 nm.

ously described for dinoflagellates (22), small amounts of Chl cwere associated with SDS/Deriphat complexes I and I' from G.polyedra. This may suggest that Chl c can be associated with aLHC associated with PSI, as has been found to be the case withChl b in Chl b-containing green plants and algae (2). Comparedto the P700-complexes, the SDS/Deriphat complexes I and I'were also enriched in yellow xanthophylls. No PSI Chl a-proteincomplex or spectral analog contained detectable amounts ofperidinin. Reelectrophoresis of the comparable Deriphat com-plex I from Glenodinium sp. onto nondenaturing SDS gels hasgenerated a high mol wt Chl a-protein complex with electropho-retic and spectral characteristics identical to those of the P700-Chl a-protein complex originally isolated from this dinoflagellateby Prezelin and Alberte (22) (BA Boczar, BB Prezelin, unpub-lished data). All SDS/Deriphat complexes I and I' isolated fromG. polyedra and Glenodinium sp. contained polypeptides in themol wt range of 60 to 70 kD, where the dominant PSI polypep-tides occur (30).Major light-harvesting Chl a-Chl c-protein complexes have

been isolated from diverse algal groups, including brown ma-croalgae (2-4), cryptomonads (I1), diatoms (1, 18) and dino-flagellates (5-7). Complexes II and II' from Gonyaulax polyedrahad electrophoretic mobilities and spectral properties similar tothe dinoflagellate Chl a-Chl c-protein complex first isolated fromGlenodinium sp. These complexes all contained a large portion

33 57 45224~~~~~~~~~~3

IV W~~~~~~~~~~~~~~~~~~~~~I

of cellular Chl c (40->80%) and had consistently low Chl acmolar ratios (0.26-0.33). Devoid of peridinin, but containingsome yellow xanthophylls, it is clear these Chl a-Chl c-proteincomplexes contribute greatly to the blue light-absorbing capabil-ities of dinoflagellates. By comparison, Chl a-Chl c-protein com-plexes from the other algal groups had higher Chl a:c molarratios, ranging from 0.5 to 4.0 (1, 3, 11, 18). The dinoflagellateChl a-Chl c complexes were first postulated by Prezelin andAlberte to be LHC situated presumably between more peripheralperidinin-Chl a-protein complexes and photosystem II imbeddedwithin the thylakoid membranes (22). While fluorescence excit-ation data presented here supports the functional role ofcomplexII as an important light-harvesting Chl a-Chl c-protein complex,its placement within the photosynthetic apparatus of dinoflagel-lates remains to be defined.Gonyaulax polyedra SDS/Deriphat complexes III, III', IV,

IV', and V all represented Chl a-Chl c-peridinin complexes andmight be considered analogous to Chl a-Chl c-fucoxanthin com-plexes found in diatoms and brown macroalgae. Of these dino-flagellate complexes, the LL G. polyedra complex III had thelowest Chl a:c ratio (1:1). All other Chl a-Chl c-peridinin com-plexes from this dinoflagellate, as well as Glenodinium sp. hadChl a:c ratios between 2.5 and 4.0. It is not known if all Chl c2associated with peridinin in G. polyedra represents distinct Chla-Chl c2-protein complexes and/or distinct Chl c2-protein (PCP)complexes which associate with the water-soluble peridinin-Chla-protein complexes, and/or if Chl c2 is associated with Chl aand peridinin in a single complex distinct from and not includingPCP complexes. Fucoxanthin-containing algae are known tocontain complexes representative of all of these chromophorearrangements ( 1-4, 8, 9, 18).

It is known that the initial fractionation procedure used hereremoves only a portion of the total cellular PCP prior to SDSsolubilization. Since no free pigment is generated on the Deriphatgels, intact PCP is presumably associated with at least one of theperidinin-containing complexes. Also, all peridinin-containingcomplexes from G. polyedra also contain protein components inthe mol wt range (32 kD) of the apoprotein of G. polyedra PCP.In addition, these complexes have spectral characteristics remi-niscent of purified PCP, which accounts for at least of 70% ofthe total peridinin in this dinoflagellate (19, 24, 25). However,immunological studies would be required to conclude that thePCP apoprotein is part of these peridinin-containing complexes.While few studies in chromophytic algae have followed alter-

ations of membrane-bound Chl a-protein complexes in responseto light intensity, Owens and Wold did document changes in the

b

FIG. 8. Laemmli gel silver stain patterns (scanned at 546nm) of polypeptides isolated from G. polyedra (a) LL (80tE m-2 s-') complexes I1I, IV, and V, and (b) HL (330 uEm-2 s_') complexes III' and IV'. Determined mol wt shownfor each polypeptide band is expressed as kD.

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BOCZAR AND PREZELIN

Chl a distribution among Chl a-protein complexes of P. tricor-nutum grown under three different light intensities (18). Thephotoadaptive responses of Glenodinium sp. and G. polyedrahave been well characterized (19, 26). Increased pigmentation inLL grown Glenodinium sp. occurs primarily through increasedcellular levels of PCP, while photoadaptation of G. polyedra tolow growth irradiance involves cellular accumulation of bothPCP and unidentified Chl a-Chl c-containing components. Inthe present study, growth irradiance wasobser-ved to affect thepigment composition of specific Chl a-protein complexes. Com-pared to high light cells, the percentage distribution of Chl c2among the complexes of low light cells changed noticeably. Thepercent gel Chl c2 associated with complex II (II') was greater inHL grown cells, while Chl c2 associated with the remainingcomplexes was reduced. These changes were different than thosedocumented in another dinoflagellate in response to HL. InGlenodinium sp., Chl c2 percentages did not change amongcomplexes, while the percent of Chl a associated with peridinindecreased (6). Other changes in the number of resolvable Chl-protein complexes and Chl distribution among them also oc-cuffed in response to changes in G. polyedra growth irradiance.Most notable was the appearance in LL cells of a fifth complex,the enrichment of Chl c in LL complex III, and the relativereduction of total Chl c associated with all peridinin-containingcomplexes in HL cells. While the present study provides somemolecular insights into the photoadaptive responses of G. pa.lyedra, future studies are needed to identify the molecular topol-ogy, photochemical function, and supramolecular organizationof these Chl a-protein complexes within the photosyntheticapparatus of dinoflagellates.

Acknowledgments-We are indebted to Drs. B. M. Sweeney and E. L. Triplettfor the use of equipment, and to Allen Matlick and Steve Passmore for technicalassistance. We would also like to thank Dr. Sweeney, Scott Newman, and ananonymous reviewer for editorial assistance.

LITERATURE CTlED

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3. BARRErr J, JM ANDERSON 1980 The P700-chlorophyll a-protein complex andtwo major light-harvesting complexes of Acrocarpia paniculata and otherbrown seaweeds. Biochim Biophys Acta 590: 309-332

4. BARRETT J, S THORNE 1981 Isolation of a F694-chlorophyll a-protein complexwith low fluorescence yield and a chlorophyll c2-protein and a fucoxanthin-protein from brown algae. In G Akoyunoglou, ed, Proc 5th Int CongrPhotosynthesis, Vol III. Balaban International Science Services, Philadelphia,pp 347-357

5. BOCZAR BA 1985 Functional organization of chlorophyll in chlorophyll c-containing marine phytoplankton. PhD thesis. University of California,Santa Barbara

6. BOCZAR BA, BB PREZELIN 1986 Light and MgC12-dependent characteristics of

four chlorophyll-protein complexes isolated from the marine dinoflageliate,Glenodinium sp. Biochim Biophys Acta 850: 300-309

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8. FRIEDMAN AL, RS ALBERTE 1984 A diatom light-harvesting pigment-proteincomplex. Purification and characterization. Plant Physiol 76: 483-489

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13. JEFFREY SW 1981 An improved thin-layer chromatographic technique formarine phytoplankton pigments. Limnol Oceanogr 26: 191-197

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16. MARKWELL J, JP THORNBER, RT BoGGs 1979 Higher plant chloroplasts:evidence that all the chlorophyll exists as chlorophyll-protein complexes.Proc Natl Acad Sci USA 76: 1233-1235

17. MORRISSEY JH 1981 Silver stain for proteins in polyacrylamide gels: a modifiedprocedure with enhanced uniform sensitivity. Anal Biochem 1I17: 307-312

18. OWENS TG, ER WOLD 1986 Light-harvesting function in the diatom Phaeo-dactylum tricornutum. 1. Isolation and characterization of pigment-proteincomplexes. Plant Physiol 80: 732-738

19. PREELIN BB 1976 The role of peridinin-chlorophyll a-proteins in the photo-synthetic light adaptation of the marine dinoflageliate, Glenodinium sp.Planta 130: 225-233

20. PREZELIN BB 1981 Light reactions in photosynthesis. In T Platt, ed, Physiolog-ical Bases of Phytoplankton Ecology. Canadian Bulletin of Fisheries andAquatic Sciences, No. 210, Ottawa, pp 1-46

21. PREZELIN BB 1986 Photosynthetic physiology of dinoflagellates. In M Taylor,ed, The Dinoflagellates. Blackwell Publications, London, pp 174-223

22. PREZELIN BB, RS ALBERTE 1978 Photosynthetic characteristics and organiza-tion of chlorophyll in marine dinoflagellates. Proc Natl Acad Sci USA 75:1801-1804

23. PREZELIN BB, BA BOCZAR 1986 Molecular bases of cell absorption andfluorescence in phytoplankton: potential applications to studies in opticaloceanography. In FE Round, DJ Chapman, eds, Progress in PhycologicalResearch, Vol 4. Biopress Ltd, Bristol, pp 349-464

24. PREZELIN BB, FT HAXO 1976 Purification and characterization ofofperidinin-chlorophyll a-proteins from the marine dinoflagellates Glenodinium sp. andGonyaulax polyedra. Planta 130: 251-256

25. PREZELIN BB, AC LEY, FT HAXO 1976 Effects of growth irradiance on thephotosynthetic action spectra of the marine dinoflagellate, Glenodinium sp.Planta 130: 251-256

26. PREZELIN BB, BM SWEENEY 1978 Photoadaptation of photosynthesis in Gon-yaulax polyedra. Mar Biol 48: 27-35

27. TANADA T 1951 The photosynthetic efficiency of carotenoid pigments inNavicula minima. Am J Bot 38: 276-290

28. THORNBER JP 1975 Chlorophyll proteins: light harvesting and reaction centercomponents of plants. Annu Rev Plant Physiol 26: 127-158

29. THORNBER JP, RS ALBERTE, FA HUNTER, JA SHIOZAWA, K-S KAN 1977 Theorganization of chlorophyll in the plant photosynthetic unit. In JM Olson,G Hind, eds, Chlorophyll-Proteins, Reaction Centers, and PhotosyntheticMembranes. Brookhaven Symp Biol 28: 132-148

30. VIERLING E, RS ALBERTE 1983 P700 Chlorophyll a-protein. Purification,characterization, and antibody preparation. Plant Physiol 72: 625-633

812 Plant Physiol. Vol. 83, 1987



chlorophyll-protein complexes from the red-tide ... · chlorophyll a, chlorophyll c2, and peridinin...

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