Light-harvesting chlorophyll c-like pigment in Prochloron

Proc. Natl. Acad. Sci. USA 

Vol. 91, pp. 679-683, January 1994 

Microbiology 

Light-harvesting chlorophyll c-like pigment in Prochloron 

(endosymbiosis/phylogeny/Prochlorophyta) 

A. W. D. LARKUM*, C. SCARAMUZZI*, G. C. Cox*, R. G. HILLERt, AND A. G. TURNERt 

*School of Biological Sciences and Electron Microscope Unit, University of Sydney, Sydney, NSW 2006, Australia; tSchool of Biological Sciences, Macquarie 

University, North Ryde, NSW 2113, Australia; and tBiological Sciences, Sydney Institute of Technology, Ultimo, NSW 2007, Australia 

Communicated by Andrew A. Benson, August 5, 1993 

ABSTRACT A chlorophyll c-like pigment, similar to magnesium-3,8-divinyl 

pheoporphyrin as monomethyl ester, has 

been isolated from Prochloron sp. obtained from five species of 

didemnid ascidians from the Great Barrier Reef, Australia, 

and from Palau, Micronesia. The pigment represents 4-15% of 

the total chlorophyll content and is shown to function in a 

light-harvesting pigment protein complex of Prochloron. The 

observation that all of the major chlorophylls (a+b+c) function 

in a light-harvesting role in Prochloron and possibly in other 

prochlorophytes is discussed in terms of the phylogeny of the 

prochlorophytes. 

The discovery of Prochloron sp., a symbiotic prokaryotic 

alga with chlorophyll (Chl) b and Chl a (1, 2), stimulated 

debate as to the origin of chloroplasts. Discussion has continued 

to the present time, fueled by the recent discoveries of 

two other prochlorophytes-the filamentous freshwater alga 

Prochlorothrix hollandica (3) and the small, unicellular, 

free-living planktonic alga Prochlorococcus marinus (4). 

Some view the prochlorophytes as allied to the ancestors of 

green chloroplasts (4, 5), while others consider them as 

members of the cyanobacteria, which have lost phycobiliproteins 

and gained Chl b, probably independently, in each 

group (6-10). The presence of a Chl c-like pigment, tentatively 

identified as magnesium 3,8-divinylphaeoporphyrin a5 

monomethyl ester (MgDVP), has been claimed for Prochlorococcus 

(4, 11), although only the peak wavelengths of the 

natural and demetallated pigment were provided (11). It also 

has been claimed that MgDVP occurs in Prochlorothrix 

hollandica (11) in low concentrations. Here we present 

evidence, based on the spectra of pigments resolved by 

thin-layer chromatography (TLC) and high-performance liquid 

chromatography (HPLC), that a Chl c-like pigment tentatively 

identified as MgDVP occurs in Prochloron in concentrations 

that are 4-15% of the total Chl. Moreover, the 

pigment is shown to be located in a light-harvesting Chl 

pigment-protein complex (LHC) and to act as a lightharvesting 

pigment. 

MATERIALS AND METHODS 

Prochloron spp. were isolated from five didemnid ascidians 

(Table 1) at One Tree Reef, Capricorn Section, Great Barrier 

Reef, as described (15). 

Analysis of the pigments ofProchloron was done on site by 

reverse-phase HPLC using a Waters 600E quaternary pump 

system with a Waters 990 photodiode array detector and a 

Shimadzu RF-551 spectrofluorimeter. The reverse-phase 

system was used with a C18 column (3.9 x 150 mm Waters 

Novapak 36975; 4-,um particle size, spherical end-capped 

material with a low carbon load), and gradient elution with 

acetonitrile/methanol/water (method 1) was applied as fol- 

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679 

lows: initial conditions, 95% solvent A/5% solvent B; linear 

gradient to 1% A/99% B in 15 min; and then held at 1% 

A/99%o B for 15 min [solvent A = 50% water/25% acetonitrile/25% 

methanol (vol/vol); solvent B = 50% acetonitrile/ 

50% methanol (vol/vol); flow rate, 2 ml min-1]. 

Further analyses used (i) TLC with polyethylene (Super 

Rylan 7007, sample no. PRX 1013, Koppers, Warrington, PA) 

and with acetone as the mobile phase or (ii) the same 

polyethylene used in an HPLC system (16) (hand-packed 4.6 

x 250 mm steel column) with a step solvent system (method 

2) as follows: system preequilibrated with 65% acetone/35% 

water; step gradient to 100% acetone at 0 min and continued 

for 15 min; flow rate, 2 ml min-1. 

Eluant from the Chl peaks separated by HPLC was collected, 

frozen, and freeze-dried under vacuum. Pigments 

were redissolved in 90% acetone/10% water, 100% acetone, 

or 100% ether, and their spectra were analyzed in a Beckman 

DU-8 spectrophotometer. Chl ratios were determined on site 

in 90% acetone/10% water by using an Hitachi U-2000 

spectrophotometer and the trichromatic equation of Jeffrey 

and Humphrey (13). Demetallated Chl (i.e., removal of Mg 

from the porphyrin ring) was produced by adding 50 ,4 of 0.1 

M HCl to 2-4 ml of acetone or ether extracts. The LHC of 

Prochloron was isolated as described (15). Low-temperature 

(77 K) fluorescence spectra were measured with a Perkin 

Elmer 44B Fluorimeter. 

Micromonas pusilla (culture CS-86 from the Culture Collection, 

CSIRO Fisheries, Hobart, Tasmania) was grown in 

GP medium (17) under natural but low light (maximum, 200 

,uE-m-2s-1). 

RESULTS AND DISCUSSION 

Analysis by HPLC in the field of the 90% acetone extracts of 

each of five isolates revealed the presence of a chlorophyll 

c-like pigment (Table 1) that was eluted at 14 min; Chl a, Chl 

b, and carotenoids were also present. A typical elution profile 

and an on-line spectrum of the Chl c-like pigment are shown 

in Fig. 1. Comparisons (not shown) of the extracts from 

Prochloron with a brown alga (possessing Chl cl + c2) and a 

dinoflagellate (Chl c2) indicated that the HPLC system separated 

the Chl c-like pigment from Chl c1 and Chl c2, which 

themselves were partially separable from each other. 

The use of HPLC in the field was crucial for the discovery 

of the Chl c-like pigment(s) in Prochloron. At present 

Prochloron cannot be cultured (18), and didemnid ascidians, 

the major source of material, release acids when handled. 

Thus, unless fresh material is extracted and processed carefully 

and immediately, Chl breakdown products (e.g., chlorophyllides 

and pheoporphyrins) can be generated, which 

would obscure the presence of Chl c-like pigments. However, 

further laboratory work was necessary, using polyethylene 

chromatography (14, 16, 19), to verify the presence of Chl 

Abbreviations: Chl, chlorophyll; BChl, bacteriochlorophyll; LHC, 

light-harvesting Chl pigment-protein complex; MgDVP, magnesium 

3,8-divinylphaeoporphyrin a5 monomethyl ester.

680 Microbiology: Larkum et al. 

Table 1. The ratios of Chl a/b- and Chl a/c-like pigment found 

by reverse-phase HPLC analysis (method 1) for Prochloron sp. 

from five didemnid ascidians collected at One Tree Reef, 

Capricorn Section, Great Barrier Reef 

Species of ascidian host for Prochloron sp. Chl a/b Chl a/c-like 

Didemnum molle (Herdmann, 1886) 3.1 15.8 

Diplosoma virens (Hartmeyer, 1908) 2.9 5.0 

Lissoclinum patella (Gottschaldt, 1898) 3.1 6.2 

Lissoclinum bistratum (Sluiter, 1905) 7.6 9.6 

Trididemnum paracyclops (Kott, 1980) 3.55 6.1 

Quantitation based on absorbance of individual peaks and extinction 

coefficients in methanol (12). Note that 90% acetone/10%o water 

extracts of the symbionts gave similar values for the Chl a/b ratio 

and, surprisingly, also for the Chl a/c-like ratio (13), despite the 

difference in absorption maximum of Chl c and MgDVP in the red 

region of the spectrum (14). 

c-like pigments. Thus, carefully prepared frozen or freezedried 

samples of Prochloron from L. patella were further 

analyzed in Sydney (the frozen samples came from a site at 

15-m depth near Lizard Island, Great Barrier Reef; the 

freeze-dried samples were a gift to G.C.C. from R. Lewin and 

were collected in Palau, Micronesia). TLC analysis (14, 19) 

revealed the presence of two Chl c-like compounds that had 

a significantly greater Rf value than those of either Chl c1 or 

Chl c2 (Fig. 2) and migrated in the relative position ofMgDVP 

(14, 19). There is also evidence from the fluorescence spectra 

of two related pigments (see below). The presence of a Chl 

c-like pigment was also confirmed by reverse-phase HPLC 

with a polyethylene column, but only one broad peak was 

resolved (Fig. 3). The spectral peaks of the compound(s) 

eluted at 7.9 min (Fig. 3 Lower, solid curve) (438 nm, 576 nm, 

and 626 nm-in 90% acetone/10% water) agree well with 

those of MgDVP (14, 19) in the same solvent. With extracts 

of brown algae or dinoflagellates, Chl c1 or Chl c2 or both 

were eluted later (not shown). The spectral peaks of the 

demetallated Chl c-like pigment (Table 2) (419 nm, 568 nm, 

and 588 nm) match well with those of demetallated MgDVP 

(11, 20). In addition, cells ofM. pusilla were extracted in 90% 

0 

0 

.0 

0 

.0 

0.2 

ol 1 

Time, min 

300 500 

Wavelength, nm 

700 

FIG. 1. (Upper) An elution profile (Adet = 440 mm) of the 90%o 

acetone extract of Prochloron sp. from Diplosoma virens separated 

by reverse-phase HPLC (method 1; see Table 1) on a Novapak C18 

column (Table 1). Bands: a, Chl a; b, Chl b; c, Chl c-like pigment; 

others, carotenoids. (Lower) On-line spectrum of Chl c-like pigment 

eluted as band c at 14.4 min. [This band was also detected by 

fluorescence emission in the red region of the spectrum (excitation, 

440 nm) with a maximum at 635 nm (not shown).] 

0 

co 

0 .0 

Q 

Proc. Natl. Acad. Sci. USA 91 (1994) 

O carotenoids 

* phaeophytins 

0 chi a(±b) 

@ chl c-like 

e chl c-like 

* chl c1 

0 chl c2 

* Origin 

A B C D E F 

o' 

. 0 

0 0 @ 0 

* 0 0 0 - - 

% a) 440 nm 

.07 . b)438 nm 

.05 

.05 .04 a 

.03b .03 

I 1 500 600 


FIG. 2. (Upper) Diagram of a developed thin-layer chromatogram 

(14) of the following 90% acetone extracts. Lanes: A, Endarachne 

binghamiae (Phaeophyta); B, Colpomenia sinuosa (Phaeophyta); C, 

Prochloron sp. (L. patella from Palau); D, Prochloron sp. (L. patella 

from Lizard Island); E, Pavlova lutheri (Prymnesiophyta); F, Amphidinium 

carterae (Pyrrophyta). The developing solvent was acetone. 

In lane C only the lower Chl c-like pigment was clearly visible, 

since the upper Chl c-like pigment was overlain by an orange 

pigment, probably a carotenoid. (Lower) Spectra of two Chl c-like 

pigments in 90% acetone from lane D in Upper. Spectra: a, spectrum 

of lower Chl c-like (stippled) pigment; b, spectrum ofupper Chl c-like 

(hatched) pigment. (Amax for the major peak of a and b spectra are 

shown.) 

acetone/10% water and analyzed by HPLC with a C18 

column (method 1). A Chl c-like pigment was present, which 

was coeluted with the major Chl c-like pigment from Prochloron. 

It also had similar spectral properties to the Chl c-like 

pigment of Prochloron (Table 2). The Chl c-like pigment in 

Micromonas spp. has been tentatively identified as MgDVP 

(12, 14, 19) on the basis of its spectral properties and elution 

characteristics on TLC and HPLC. 

MgDVP has a fully unsaturated porphyrin macrocycle and 

does not carry a long-chain esterifying alcohol at C-17 and 

differs from Chl c2 only in the presence at C-17 of a propionic 

acid instead of an acrylic acid (14). Evidence suggests that 

MgDVP is an intermediate in the pathway of Chl a [and 

bacteriochlorophyll (BChl)] biosynthesis (21). It was first 

documented in the photosynthetic bacterium Rhodobacter 

sphaeroides grown in the presence of 8-hydroxyquinoline, 

but it probably occurs also in some mutants ofR. sphaeroides 

as a result of a lesion in the normal synthesis of BChl (21). 

MgDVP was also identified as a pigment ofunknown function 

in several marine flagellates (22) including the prasinophyte 

M. pusilla, where it has since been shown to be present in an 

LHC as a light-harvesting pigment (23, 24). However, it 

should be pointed out that the precise structure of the Chl 

c-like pigment in M. pusilla and the related alga Mantoniella 

squamata is debated (12, 20) [both algae are placed in the 

special group, the micromonadophytes (25)]. MgDVP is also 

probably present in the prochlorophyte Prochlorococcus 

marinus (11), although full details have yet to be documented. 

On the basis of the present evidence, we conclude that a chl 

c-like pigment, similar to MgDVP, occurs in Prochloron sp. 

together with a closely related pigment; however, both pigments 

need to be further examined by nuclear magnetic

0 

c 

0. 

co °0.1 

CO 0.15 

Microbiology: Larkum et al. 

0.1 

0.05 

0 2 4 6 

Time, min 

a 

573 


8 12 

FIG. 3. (Upper) Profile of pigments extracted in 90%o acetone 

from Prochloron sp. (L. patella from Palau) and separated by 

reverse-phase HPLC on a polyethylene column (17) (method 2) 

monitored at 440 nm (the eluant was also monitored by a fluorescence 

detector, which confirmed the fluorescence of Chl c-like at 635 nm 

in the band eluted at 7.9 min). Bands: a, Chl a + b; c, Chl c-like; 

others, carotenoids. Extracts from a brown alga E. binghamiae and 

a dinoflagellate alga A. carterae showed that Chl ci was eluted at 9.2 

min and Chl c2 was eluted at 11.8 min. (profiles not shown). (Lower) 

On-line spectrum of the Chl c-like band (c) eluted at 7.9 min. (solid 

curve) and of the pigment extracted and chromatographed in the 

same position from the LHC (dashed curve). 

resonance spectroscopy. It is possible that only one of the 

pigments is native and that the other is a product of the 

preparative procedures. We reject the possibility that both 

pigments are the result of contamination by another alga or 

photosynthetic bacterium. The preparations used were monitored 

microscopically and showed less than 0.3% contamination 

on a cell count basis (

682 Microbiology: Larkum et al. 

U) 

0 657 

a: 

600 ~~720 

_ 

636 628 

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 

A, nm 

FIG. 4. Low-temperature (77 K) fluorescence emission spectra of the LHC isolated from Prochloron sp. (from L. patella found at 10-m depth 

on One Tree Reef). (A) Isolated LHC excited by light at 440 nm. (B) LHC extract in 95% acetone/10% water excited at 440 nm. (C) LHC extract 

in 95% acetone/5% water excited at 470 nm. (D) LHC in 2% SDS and heated to 70°C for 5 min, excited at 440 nm. (E) LHC in 2% SDS and 

heated to 70°C for 5 min, excited at 470 nm. 

procedure (15) as the LHC had no detectable levels of the Chl 

c-like pigment, although Chl a and b were present, as shown 

previously (15). This evidence suggests that the Chl c-like 

pigment is not intimately connected to photosystem I. The 

nature and function of the major LHC in Prochloron sp. and 

the presence of other LHCs have yet to be fully elucidated. 

It may be similar to LHC II of chlorophytes and higher plants 

and therefore associated with photosystem II (15), but neither 

its molecular location nor its primary structure has been 

reported. 

The occurrence of Chls a, b, and c in a light-harvesting role 

in Prochloron raises many intriguing questions. The presence 

of an identical or similar Chl c-like pigment to that in 

Prochloron seems likely in Prochlorococcus marinus, although 

it has yet to be shown in the latter organism whether 

the pigment acts in a light-harvesting capacity and is found in 

an LHC. Is Prochlorothrix hollandica, the other prochlorophyte, 

similar? Preliminary pigment analysis of that organism 

indicates that MgDVP may be present in low concentrations 

(11). If this is substantiated, then all three prochlorophytes 

are likely to contain the Chl c-like pigment, although it would 

still be necessary to show that it acts in a light-harvesting 

capacity. At the moment it is possible to say that Prochloron 

sp. and possibly each of the three prochlorophytes are more 

closely allied to the micromonadophyte chloroplast line (25, 

28) than to any other chloroplast line on the evidence that 

they all share a Chl c-like pigment. However, the distinct 

differences in the LHCs between Prochloron (15) and 

Prochlorothrix (29) on the one hand (the type of LHC in 

Prochlorococcus is not known) and the micromonadophytes 

on the other hand (20, 30) (where the LHC is of the chromophytic 

type) stand against a close affinity between these 

two groups. The recent finding of divinyl Chl a and b in 

Prochlorococcus (4, 11) and the evidence that both Prochloron 

and Prochlorococcus share the presence of a Chl c-like 

pigment raise the possibility that divinyl chlorophylls a and b 

also exist in Prochloron. 

The occurrence of a Chl c-like pigment and Chl b [or 

divinyl-Chl b (11)] in all of the prochlorophytes would 

weaken the argument that these organisms are merely cyanobacteria, 

where Chl b originated independently in each of 

the three groups (7-10). We believe that the phylogenetic tree 

analysis on which that argument was based is flawed (31-33). 

Another explanation is that the prochlorophytes are an 

anciently diverged group (34) widely separated from the 

Proc. Natl. Acad. Sci. USA 91 (1994) 

Cyanobacteria, as originally suggested (2). Thus, all three 

known prochlorophytes may be only very distantly related to 

the green chloroplast and may have diverged considerably 

from each other. Finally, the present evidence lends support 

to the recent suggestion that Chl c-like pigments were present 

in the earliest photosynthetic prokaryotes (28). 

We are grateful to Dr. S. W. Jeffrey for the supply of M. pusilla. 

This work was supported by the Australian Research Council (Small 

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Light-harvesting chlorophyll c-like pigment in Prochloron

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