Plant Physiol. (1989) 91, 727-7320032-0889/89/91/0727/06/$01 .00/0

Received for publication December 5, 1988and in revised form June 18, 1989

A New Form of Chlorophyll c Involved in Light-Harvesting1

Marvin W. Fawley

Department of Botany, North Dakota State University, Fargo, North Dakota 58105

ABSTRACT

A new form of chlorophyll c has been isolated from the pyrm-

nesiophyte Paviova gyrans Butcher. This pigment is spectrallysimilar to chlorophyll c2, but all the absorption maxima (454, 583,and 630 nm in diethyl ether) are shifted 4 to 6 nanometers tolonger wavelengths. The new pigment can be separated fromother chlorophyll c-type pigments by reversed-phase high per-

formance liquid chromatography and thin layer chromatography.Both chlorophylls cl and c2 are found with the new chlorophyll c

pigment in P. gyrans, and it has also been detected in thechrysophyte Synura petersenii Korsh. The light-harvesting func-tion of the new chlorophyll c pigment is indicated by its presencealong with chlorophyll c1 and c2 in a light-harvesting pigment-protein complex isolated from P. gyrans in which chlorophyll c

pigments efficiently transfer absorbed light energy to chlorophylla.

The Chl c pigments are widely distributed among marineand freshwater algae. These Chl forms differ from both Chl a

and Chl b in that ring IV is unsaturated and there is a lack ofa hydrophobic esterified phytol side chain. This combinationof characteristics is also found in the protochlorophyllides,and Chl c forms have been called chlorophyllides c (27).Originally, two forms of Chl c were recognized, both ofwhichhave acrylic acid groups at C7 (15). Chl cl has an ethyl group

present at position C4 in ring II ofthe Chl macrocycle, whereasChl c2 has a vinyl group in this position (4, 25). This differenceis not sufficient to enable separation in most conventionalchromatographic systems, such as cellulose TLC or RP2-HPLC. A polyethylene-based TLC separation system was usedby Jeffrey ( 15) to determine that Chl cl and c2 occur togetherin chrysophytes, diatoms, brown algae, and a few dinoflagel-lates, while most dinoflagellates and cryptomonadspossess only Chl C2 (17). Where studied, both ofthese pigmentshave been shown to be involved in light-harvesting. Chrom-ophytes in which light-harvesting pigment-protein complexescontaining some form of Chl c have been isolated includebrown algae (2), diatoms (6, 10, 22), cryptomonads (14),dinoflagellates (3), prymnesiophytes (7, 13), and a chryso-phyte (28).

Jeffrey and Wright (20) and Vesk and Jeffrey (26) haverecently isolated and spectrally characterized a third Chl c

species, which they called Chl C3. This pigment migrated withChl c2 in the polyethylene TLC system, but could be separated

' Supported by National Science Foundation EPSCoR grantR1186 10675.

2 Abbreviation: RP. reversed-phase.

from both Chl cl and c2 by reversed-phase high performanceTLC. Chl C3 has also been separated from Chl cl and c2 byRP-HPLC (9). Chl C3 is also implicated in light-harvesting inthe prymnesiophyte Emiliania huxleyi, since the Chl c presentin this alga has been shown to contribute to light harvesting(12). Stauber and Jeffrey (24) recently found several diatomswith Chl C3, and it is likely that it is to be found in moreorganisms.Another pigment which is structurally very similar to Chl

cl and Chl c2 is magnesium 2,4-divinylpheoporphyrin a5monomethyl ester, which was initially isolated from a pho-tosynthetic bacterium (21). This pigment is also found inseveral small green flagellates (Micromonadophyceae) (23),although the actual identity of the Chl c-like pigment in theseorganisms has been questioned (29). In these green algae, Mg2,4-divinylpheoporphyrin a5 monomethyl ester is present ina light-harvesting complex which also contains Chl a and Chlb (8, 30). In Mg-2,4-divinyl-pheoporphyrin a5 monomethylester, the acrylic acid group at C7 is replaced by a proprionicacid group.

I recently (5) described a RP-HPLC system for the separa-tion of pigments from the prymnesiophyte Pavlova gyrans.Using that procedure, it appeared that Chl cl and Chl c2 werebeing separated. However, in trying to extend that procedureto other organisms, it soon became apparent that Chl c, andChl c2 were not being separated. Rather, a new form of Chl cwith spectral characteristics similar to Chl c2 is present in P.gyrans. Here I describe the characteristics and function of thisnew Chl c pigment and compare it to previously describedforms of Chl c.

MATERIALS AND METHODS

Culture Methods

Pavlova gyrans Butcher (Culture Collection of Marine Phy-toplankton, Bigelow Laboratory for Ocean Sciences, WestBoothbay Harbor, ME (CCMP), clone MPPAV; Phaeodac-tylum tricornutum Bohlin (University of Texas at AustinCulture Collection of Algae [UTEX] 646); Mantoniella squa-mata (Manton & Park) Desikachary (UTEX 990), and Prym-nesium parvum Carter (CCMP clone PRYM) were grown inmodified ES enriched artificial seawater medium (ESAW) (7)and Synura petersenii Korsch. UTEX 845 was grown inmodified Woods Hole freshwater medium (8). Cultures of allorganisms were maintained in one to 2 L Erlenmeyer flasks.Marine organisms were continually bubbled with air, whileS. petersenii was not. Temperature was constant at 20°C, andillumination was provided by cool-white fluorescent lights atabout 50 ME/m2/s with a 14 h: 10 h light:dark cycle.

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Pigment Extraction

Algae were harvested from dense, late-log phase cultures bycentrifugation and pigments were extracted using three meth-ods: (a) extraction of the cell pellet with at least nine volumesof ice-cold 100% acetone; (b) extraction of the cell pellet withat least nine volumes of ice-cold 100% methanol; and (c)freezing the pellet with liquid nitrogen, followed by thawingthe pellet in at least nine volumes of 100% acetone. Duringextraction, all procedures were performed on ice. For HPLC,pigment extracts were centrifuged, and 20 to 100 ,uL ofsupernatant was injected into the HPLC system. For TLC,the pigments were transferred to fresh anhydrous diethyl etherbefore application.

HPLC

The HPLC system used was that described previously (5).This is a RP system consisting of an Alltech Econosphere 5,um C18 cartridge column (25 cm x 4.6 mm). The solventsystem used was B) 50% methanol, 50% acetonitrile (v/v); A)50% water, 50% B (v/v). Pigments were eluted from thecolumn using a linear gradient of 0 to 80% B in 10 min,followed by a linear gradient from 80 to 98% B in 10 min,with continued elution with 98% B until all pigments wereremoved from the column. The initial flow rate of 1.5 ml/min was changed to 2.5 ml/min at 30 min. Fractions werecollected as they eluted from the column.

TLC

Cellulose TLC to separate Chl c pigments from all otherpigments was performed by the method ofJeffrey (18), exceptthat commercial plates (EM Science No. 5577) were used.Polyethylene (Polysciences No. 2719) TLC to separate Chl clfrom c2 was performed using the method of Jeffrey (16),except that the adsorbent layer was 250 um and the solventwas 100% acetone.

Spectral Characterization

Pigments separated by HPLC were transferred from theelution solvent to diethyl ether, and those purified by TLCwere eluted directly into the appropriate solvent. Absorptionspectra were recorded using a Beckman DU-7 spectrophotom-eter. Magnesium-free derivatives were prepared in acetone bythe method of Jeffrey and Shibata (19).

Isolation of P. gyrans Light-Harvesting Complex

A light-harvesting pigment-protein complex was isolatedfrom P. gyrans by the method of Fawley et al. (7). Pigmentswere extracted in methanol as described above.

RESULTS

Two fractions containing Chl c pigments were separated byRP-HPLC of pigment extracts from Pavlova gyrans (Fig. IA).As previously reported (5), the first fraction had spectralcharacteristics similar to Chl cl, and the second, Chl c2.However, RP-HPLC of pigment extracts of Phaeodactylum

C)z.4

com0(0)

4

0 10 20 30 40 50TIME (minutes)

Figure 1. Separation of pigments from extracts of (A) Pavlova gyransand (B) Phaeodactylum tricornutum by RP-HPLC. P. gyrans pigmentswere extracted from pelleted cells with at least nine volumes of ice-cold acetone. P. tricomutum pigments were extracted from the cellpellet with at least nine volumes of ice-cold methanol. Followingcentrifugation, 20 AL samples were injected into the HPLC systemdescribed in the text. Detection was at A440 nm, 0.16 full scale. Peakidentities: 1, Chl cl and ChI c2; 2, new Chl c pigment; 3, fucoxanthin;4, diadinoxanthin; 5, diatoxanthin; 6, Chi a; 7, ,B-carotene.

tricornutum produced only a single Chl c fraction (Fig. 1B).Since P. tricornutum is known to possess both Chl c, and Chlc2 (25), it was apparent that the fraction from P. gyrans whichhad been identified as Chl c2 was in fact a previously unde-scribed pigment. One potential concern was that this newpigment might be a chromatographic artifact. To eliminatethis possibility, fraction 1 and fraction 2 were isolated by RP-HPLC and subjected to a second run with the RP-HPLCsystem. Rechromatography of fraction 1 did not result in theproduction of fraction two, and no Chl cl or c2 resulted whenfraction 2 was rechromatographed (data not shown).To better characterize this pigment, it was compared to all

other known forms of Chl c. Chl c, and Chl c2 were isolatedby polyethylene TLC from P. tricornutum (16), Chl c3 wasisolated from the prymnesiophyte Prymnesium parvum (9) byRP-HPLC, and Mg-2,4-divinylpheoporphyrin a5 mono-methyl ester from the micromonadophyte Mantoniella squa-mata (23) by RP-HPLC and cellulose TLC.

Figure 2 shows the absorption spectra, in diethyl ether, ofthe new Chl c pigment from P. gyrans, Chl c, and c2 from P.tricornutum, Chl C3 from P. parvum, and Mg-2,4-divinylpheo-

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NEW FORM OF CHLOROPHYLL c

454

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400 51 500 600WAVELENGTH (nm)

porphyrin a5 monomethyl ester from M. squamata. Maximaand band ratios of these spectra are presented in Table I.Values are in close agreement with published results (15, 16,20, 24). The new Chl c form is similar to Chl c2, in that theratio of band II to band I is about 1; however, the absorptionmaxima of the two pigments are different by 4 to 6 nm. Themost notable difference between the new pigment and otherChl c forms is the Soret maximum, which at 454 nm indiethyl ether is the longest wavelength Soret maximum ofanyof these pigments.

Differences among the Chl c pigments are also evident inretention characteristics using different chromatographic sep-aration systems (Table II). Three systems were used: celluloseTLC, polyethylene TLC, and RP-HPLC. With the three sys-tems, Chl cl and c2 migrate identically except in polyethyleneTLC, where Chl cl migrates further than Chl c2. Chl c3 can beseparated from Chl c, and c2 by RP-HPLC, but all threepigments have the same RF values in cellulose TLC, and ChlC3 and c2 have the same RF values in polyethylene TLC. Mg-

Table I. Spectral Characteristics of Chl c Pigments in Diethyl EtherAll spectra at room temperature.

Absorption Band RatiosAlga Pigmenta MaiabAlgaPinMaxima Soret:l 11:1

nm

Paviova gyrans c* 454,583,630 13.0 1.07Pav/ova gyrans c1 444, 577, 626 8.7 0.66Paviova gyrans C2 447,580,627 13.2 1.16Phaeodactylum tricornutum c, 444, 577, 626 8.7 0.64Phaeodactylum tricornutum C2 448, 579, 627 12.1 0.99Prymnesium parvum C3 452, 585, 625 34.0 3.67Mantoniella squamata MgDV 437, 574, 624 10.0 0.52

a Chl c* = new Chl c pigment, MgDV = magnesium-2,4-divinyl-phaeoporphyrin a5 monomethyl ester. b Maxima are rounded tonearest integer.

Figure 2. Visible absorption spectra of Chl c pig-ments in diethyl ether. Insets show detail of redregion. A, new Chl c from P. gyrans; B, Chl c1 fromP. tricornutum; C, Chl c2 from P. tricornutum; D, ChlC3 from P. parvum; and E, magnesium-2,4-divinyl-phaeoporphyrin a5 monomethyl ester from M. squa-mata.

Table II. Retention of Chl c Pigments in Different ChromatographySystems

Pigmenta Cellulose Polyethylene RP-HPLCPigment9 TLC (RF) TLC (RF) (Rt, min)

Chl c* 0.07 0.80 13.9Chi c1 0.17 0.66 8.5Chl c2 0.17 0.47 8.5Chl C3 0.16 0.50 7.2MgDV 0.15 NDb 8.5

a Chl c = new Chl c pigment from P. gyrans, MgDV = magnesium-2,4-divinylphaeoporphyrin a5 monomethyl ester from M. squamata.Chl c1 and c2 were isolated from P. tricornutum and Chl C3 from P.parvum. b ND = not determined.

2,4-divinyl pheoporphyrin a5 monomethyl ester has the sameretention time (R,) as Chl cl and c2 in RP-HPLC and thesame RF as Chl cl, c2, and C3 in cellulose TLC. PolyethyleneTLC of Mg-2,4-divinylpheoporphyrin a5 monomethyl esterwas not performed, but Jeffrey and Wright (20) indicate thatthis pigment migrates faster than Chl cl in polyethylene TLC.The new Chl c pigment can be separated from other Chl cpigments by all three methods. It has a longer R, in RP-HPLCthan any of the other Chl c pigments, has a lower RF value incellulose TLC than any of the other pigments, and a higherRF than Chl cl in polyethylene TLC.The absorption spectrum of the new Chl c pigment in

acetone and also of the Mg-free derivative (pheophytin) inacetone are presented in Figure 3. Table III lists the absorptionmaxima of all the Chl c pigments and Mg-free derivatives inacetone for comparison. The spectra of the new Chl c pigmentonce again differ from those of other Chl c pigments in thatthe Soret maximum is at longer wavelength. The Mg-freederivative of the new pigment (Fig. 3) exhibits features gen-erally attributed to porphyrins, namely four maxima, a lowabsorption for band I, and a shift of about 20 nm with thetransition from Chl to pheophytin (1 1).

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Plant Physiol. Vol. 91,1989

:C.)z.4

0com4

WAVELENGTH (nm)

Figure 3. Visible absorption spectra in acetone of the new Chi cpigment ( ) and its Mg-free derivative (-----).

Table Ill. Spectral Characteristics of Chl c Pigments and Their Mg-Free Derivatives in Acetone

All spectra at room temperature.Band Ratios

Pigmenta Absorption MaximabSoret:l 11:1

nmc* 453,583,632 11.43 1.00c*-Mg 434,534,576,597 17.83 0.92Ci 447,579, 629 7.82 0.64c,-Mg 428,531,574,589 20.39 1.83c2 450,581,629 11.16 1.04c2-Mg 431,533,575,594 13.33 1.10C3 452,585,627 29.58 3.22c3-Mg 436,531,600,657 62.84 3.23MgDV 438,575,625 8.18 0.49MgDV-Mg 422, 526, 568, 593 16.63 1.36

aChl c* = new Chic pigment from P. gyrans, MgDV = magnesium2,4-divinylphaeoporphyrin a5 monomethyl ester from M. squamata.Chi cl and c2 were isolated from P. tricornutum and Chl C3 from P.parvum. b Maxima are rounded to nearest integer.

To determine the full Chl c pigment complement of P.gyrans, pigment extracts were subjected to cellulose TLC,which separated the new Chl c pigment from other Chl cpigments. The spot containing other Chl c pigments wasrechromatographed on polyethylene TLC. Two Chl c frac-tions were produced, with absorption characteristics verysimilar to Chl c, and c2 from P. tricornutum (Table I). Thenew Chl c pigment, therefore, is present with both Chl cl andc2 in P. gyrans.To determine if the new Chl c pigment functions in light-

harvesting as do other forms of Chl c, a light-harvestingcomplex known to have transfer of energy from Chl c pig-ments to Chl a (7) was isolated from P. gyrans. The isolatedcomplex was pelleted by ultracentrifugation, and pigments

extracted with methanol. The RP-HPLC chromatogram ofthis extract is presented in Figure 4. Based on retention times(5), the pigments present included Chl cl and/or c2, the newChl c pigment, fucoxanthin, diadinoxanthin, diatoxanthin,and Chl a. Polyethylene TLC of pigments extracted from thislight-harvesting complex revealed the presence of both Chl c,and c2. This light-harvesting complex from P. gyrans thereforecontains four Chl forms and three carotenoids.One other organism has thus far been shown to possess this

new Chl c pigment. The Chrysophyte Synura petersenii waspreviously reported to possess only Chl c, (1); however, theRP-HPLC chromatogram of pigment extracts from S. peter-senii indicated a fraction with retention time identical to thenew Chl c pigment (data not shown). The identity of thisfraction as the new Chl c pigment was verified by absorptionspectroscopy (Fig. 5). The new Chl c pigment was also detectedin pigment extracts from UTEX 992, the only other clone ofP. gyrans which has been examined (data not shown).

DISCUSSION

The new Chl c pigment can be distinguished from previ-ously described forms on the basis of its apparent lowerpolarity in RP-HPLC and its longer wavelength Soret maxi-mum in either diethyl ether or acetone. The absorption bandratios of this new pigment are similar to those of Chl c2.Overall the best discriminator of the five Chl c pigmentsstudied here is the Soret maximum in diethyl ether. Mg-2,4-divinylpheoporphyrin a5 monomethyl ester has the shortestwavelength Soret maximum at 437 nm, followed by Chl c, at444 nm, Chl c2 at 448 nm, Chl c3 at 452 nm, and the newChl c pigment at 454 nm.To separate these pigments, a combination of methods

must be used. Chl C3 and the new Chl c pigment can beseparated from other Chl c pigments by RP-HPLC, but thethree remaining pigments cannot be separated by this system.Cellulose TLC will separate the new Chl c pigment from allothers, but the RF is so low that care must be taken to avoidconfusion with chlorophyllides and other derivatives whichcan appear in mishandled extractions. It would appear thatthe best method presently available to separate these pigmentswith confidence is the combination of cellulose TLC, polyeth-ylene TLC, and RP-TLC used by Jeffrey and Wright (20) andStauber and Jeffrey (24). However, RP-HPLC provides anexcellent first method to determine if either Chl c3 or the newChl c pigment is present. The TLC separation system(s)needed to determine the presence of other Chl c forms canthen be chosen.The presence of Chl c alteration products in pigment ex-

tracts have been noted (15, 16); however, none of the altera-tion products have the characteristics ofthe pigment describedhere. To minimize the possibility that the new Chl c pigmentmight be an artifact that had not previously been character-ized, three pigment extraction procedures were used. The newChl c pigment was present in all of these extracts, as deter-mined by RP-HPLC. One ofthese procedures, extraction withacetone on ice, did produce some alteration products fromboth P. tricornutum and P. parvum, but not from P. gyrans.In addition, rechromatography by RP-HPLC of both Chl cfractions from P. gyrans did not result in any additional Chl

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NEW FORM OF CHLOROPHYLL c

40

Figure 4. Separation of pigments from a light-har-vesting complex of P. gyrans by RP-HPLC. Isolatedlight-harvesting complex was extracted with at leastnine volumes of ice-cold methanol and 30 uL in-jected into the HPLC system described in the text.Detection was at A440,n, 0.16 full scale. Peak iden-tities as given in Figure 1.

50

TIME (minutes)

WAVELENGTH (nm)

Figure 5. Visible absorption spectrum in diethyl ether of the new Chic pigment isolated by RP-HPLC from pigment extracts of S. peter-senii.

c peaks, indicating that the new pigment was not an artifactof the separation system.That the new Chl c pigment is able to function in light-

harvesting is strongly suggested by its presence in a light-harvesting complex which is known to have transfer ofenergyfrom Chl c pigments to Chl a, as evidenced by fluorescenceexcitation spectra and a lack of Chl c fluorescence emission(7). These fluorescence data indicate that light energy ab-sorbed by all the Chl c pigments is transferred to Chl a;however, it is not possible to separate the individual forms ofChl c to be certain the new form is actually transferring energy.The new Chl c pigment, or one of the other forms of Chl c

could be inactive. This seems very unlikely, since no fluores-cence attributable to Chl c forms is detected in the fluores-cence emission spectrum (7), indicating that all the Chl c iscoupled to Chl a. Another possibility is that the new Chl cpigment is not fluorescent in this complex, but Chl pigmentsalways fluoresce or transfer energy in light-harvesting com-plexes. The most reasonable interpretation of these results isthat the new Chl c pigment is a functional light-harvestingpigment.The presence of multiple forms of Chl c in the light-

harvesting complex from P. gyrans is in keeping with thepresence of both Chl cl and c2 in a light-harvesting complexfrom the diatom P. tricornutum (22) which is homologous tothat of P. gyrans (7). However, the light-harvesting complexfrom P. gyrans is the first pigment-protein complex knownto contain four forms of Chl.The discovery of a new form of Chl c brings to five the

number of described Chl c-like pigments in eukaryotic orga-nisms. Four of these pigments are found in the chromophytealgae: Chl cl, Chl c2, Chl C3, and the new Chl c pigment. Oneis found in the Micromonadophyceae (Chlorophyta): Mg-2,4-divinylpheoporphyrin a5 monomethyl ester. It is apparentthat structural investigations of both the new form of Chl cand Chl c3 must be undertaken so that a more meaningfulsystem of nomenclature can be devised than that presently inuse, and to determine if all of these pigments should begrouped as Chl c pigments.Not only does the chemical structure of the new Chl c

pigment need to be determined, but also its distribution inthe various groups of algae. Thus far, the new Chl c pigmenthas been found in two clones of the prymnesiophyte Pavlovagyrans and one isolate of the chrysophyte Synura petersenii.S. petersenii is interesting because it had previously beendetermined that this organism contains only Chl cl (1), oneof a group of freshwater chrysophytes which are the onlyorganisms known to possess Chl cl but not Chl c2. This

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Plant Physiol. Vol. 91,1989

investigation clearly shows that at least this single isolate alsopossesses the new Chl c.

All four Chl c forms described in the Chromophyte algaehave now been detected in the Prymnesiophyceae, althoughthe combinations vary. Some prymnesiophytes have Chl cland c2 (17), others have Chl c2 and Chl C3 (20), one has Chlcl, c2, and C3 (9), and now P. gyrans has Chl cl, c2, and thenew Chl c pigment. The organization of all these forms of Chlc in the photosynthetic apparatuses of these organisms ispresently unknown except for the light-harvesting function.This should be a fertile area for more research.

LITERATURE CITED

1. Andersen RA, Mulkey TJ (1983) The occurrence of chlorophyllscl and c2 in the Chrysophyceae. J Phycol 19: 289-294

2. Barrett J, Anderson JM (1980) The P-700-chlorophyll a-proteincomplex and two major light-harvesting complexes ofAcrocar-pia paniculata and other brown seaweeds. Biochim BiophysActa 590: 309-323

3. Boczar BB, Prezelin BB, Markwell JP, Thornber JP (1980) Achlorophyll c-containing pigment-protein complex from themarine dinoflagellate, Glenodinium sp. FEBS Lett 120: 243-247

4. Budzikiewicz H, Taraz K (1971) Chlorophyll c. Tetrahedron 27:1447-1460

5. Fawley MW (1988) Separation of chlorophylls cl and c2 frompigment extracts of Pavlova gyrans by reversed-phase highperformance liquid chromatography. Plant Physiol 86: 76-78

6. Fawley MW, Grossman AR (1986) Polypeptides of a light-harvesting complex of the diatom Phaeodactylum tricornutumare synthesized in the cytoplasm ofthe cell as precursors. PlantPhysiol 81: 149-155

7. Fawley MW, Morton SJ, Stewart KD, Mattox KR (1987) Evi-dence for a common evolutionary origin of light-harvestingfucoxanthin chlorophyll a/c complexes of Pavlova gyrans(Prymnesiophyceae) and Phaeodactylum tricornutum (Bacil-lariophyceae). J Phycol 23: 377-381

8. Fawley MW, Stewart KD, Mattox KR (1986) The novel light-harvesting pigment-protein complex of Mantoniella squamata(Chlorophyta): phylogenetic implications. J Mol Evol 23: 168-176

9. Fawley MW (1989) Detection of chlorophylls cl, c2, and C3 inpigment extracts of Prymnesium parvum (Prymnesiophyceae).J Physiol (in press)

10. Friedman AL, Alberte RS (1984) A diatom light-harvesting pig-ment-protein complex. Purification and characterization. PlantPhysiol 76: 483-489

11. Goedheer JC (1966) Visible absorption and fluorescence of chlo-rophyll and its aggregates in solution. In LP Vernon, GR Seely,eds, The Chlorophylls. Academic Press, New York, pp 147-184

12. Haxo Fl (1985) Photosynthetic action spectrum of the coccol-

ithophorid, Emiliania huxleyi (Haptophyceae): 19'-hexano-yloxyfucoxanthin as antenna pigment. J Phycol 21: 282-287

13. Hiller RG, Larkum AWD, Wrench PM (1988) Chlorophyllproteins ofthe prymnesiophyte Pavlova lutherii (Droop) comb.nov.: identification of the major light-harvesting complex.Biochim Biophys Acta 932: 223-231

14. Ingram K, Hiller RG (1983) Isolation and characterization of amajor chlorophyll a/c2 light-harvesting protein from aChroomonas species (Cryptophyceae). Biochim Biophys Acta722: 310-319

15. Jeffrey SW (1969) Properties of two spectrally different compo-nents in chlorophyll c preparations. Biochim Biophys Acta177: 456-469

16. Jeffrey SW (1972) Preparation and some properties of crystallinechlorophyll c, and c2 from marine algae. Biochim Biophys Acta279: 15-33

17. Jeffrey SW (1976) The occurrence of chlorophyll cl and c2 inAlgae. J Phycol 12: 349-354

18. Jeffrey SW (1981) An improved thin-layer chromatographictechnique for marine phytoplankton pigments. Limnol Ocean-ogr26: 191-197

19. Jeffrey SW, Shibata K (1969) Some spectral characteristics ofchlorophyll c from Tridacna crocea zooxanthellae. Biol Bull(Woods Hole) 136: 54-62

20. Jeffrey SW, Wright SW (1987) A new spectrally distinct com-ponent in preparations of chlorophyll c from the micro-algaEmiliania huxleyi (Prymnesiophyceae). Biochim Biophys Acta894: 180-188

21. Jones OTG (1963) Magnesium 2,4-divinylphaeoporphyrin a5monomethyl ester, a protochlorophyll-like pigment producedby Rhodopseudomonas speroides. Biochem J 89: 182-189

22. Owens TJ, Wold ER (1986) Light-harvesting function in thediatom Phaeodactylum tricornutum. 1. Isolation and charac-terization of pigment-protein complexes. Plant Physiol 80:732-738

23. Ricketts TR (1970) The pigments of the Prasinophyceae andrelated organisms. Phytochemistry 9: 1835-1842

24. Stauber JL, Jeffrey SW (1988) Photosynthetic pigments in fifty-one species of marine diatoms. J Phycol 24: 158-172

25. Strain HH, Cope BT Jr, McDonald GN, Svec WA, Katz JJ(1971) Chlorophylls cl and c2. Phytochemistry 10: 1109-1114

26. Vesk M, Jeffrey SW (1987) Ultrastructure and pigments of twostrains of the picoplanktonic alga Pelagococcus subvirides(Chrysophyceae). J Phycol 23: 322-336

27. Wasley JWF, Scott WT, Holt AS (1970) Chlorophyllides c. CanJ Biochem 48: 376-383

28. Wiedemann I, Wilhelm C, Wild A (1983) Isolation of chloro-phyll-protein complexes and quantification of electron trans-port components in Synura petersenii and Tribonema aequale.Photosynth Res 4: 317-329

29. Wilhelm C (1987) Purification and identification of chlorophyllcl from the green alga Mantoniella squamata. Biochim BiophysActa 892: 23-29

30. Wilhelm C, Lenartz-Weiler I, Wiedemann I, Wild A (1986) Thelight-harvesting system of a Micromonas species (Prasinophy-ceae): the combination of three different chlorophyll species inone single chlorophyll-protein complex. Phycologia 25: 304-312

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