Light-harvesting chlorophyll c-like pigment in Prochloron

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
Wavelength, nm
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