Plant Cell Physiol. 46(3): 416–424 (2005)
doi:10.1093/pcp/pci054, available online at www.pcp.oupjournals.org
JSPP © 2005
Isolation of a Novel Carotenoid-rich Protein in Cyanophora paradoxa that is
Immunologically Related to the Light-harvesting Complexes of
Photosynthetic Eukaryotes
Heather M. Rissler and Dion G. Durnford 1
Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada, E3B 6E1
;
of pigment-binding proteins and their associated chromophores, which include tetrapyrroles and carotenoids. Both soluble and integral membrane proteins have evolved the function
of light harvesting, and both types are ubiquitous amongst photosynthetic organisms.
The antennae of most cyanobacteria are comprised of linear tetrapyrroles that are covalently linked to soluble proteins
and assembled into phycobilisomes (PBS). Remnants of the
cyanobacterial antennae system have persisted in several
groups of algae including the rhodophytes and glaucophytes,
which contain fully assembled PBS, and the cryptomonads,
which possess phycobiliproteins. A second type of independently evolved light-harvesting antennae, the soluble peridinin–
chlorophyll-binding protein (PCP), is present in many dinoflagellates (Prezelin and Haxo 1976). The most widespread class of
antennae proteins found in algae and higher plants are the lightharvesting complexes (LHCs), which have three membranespanning regions (MSRs) that non-covalently bind chlorophylls
(Chls) and carotenoids (Green and Durnford 1996). Evolution
of the LHC gene family has produced an impressive array of
apoproteins that can bind a range of accessory pigments, enabling photosynthetic organisms to optimize light utilization in
diverse environments (Durnford et al. 1999).
While light harvesting is an important function of pigment-binding proteins, many photoprotective proteins have
also evolved independently. In particular, many members of the
LHC superfamily are hypothesized to serve a photoprotective
function and can be categorized based on the number of predicted MSRs. The high light-inducible proteins (HLIPs) in
cyanobacteria possess one MSR (Bhaya et al. 2002), and HLIP
homologs are also found in eukaryotes (reviewed in Heddad
and Adamska 2002). Stress-enhanced proteins (SEPs), which
posses two MSRs, are induced in response to light stress in
Arabidopsis thaliana (Heddad and Adamska 2000). The family of early light-induced proteins (ELIPs) possess three MSRs
and are important in mediating defense against photo-oxidative stress (Hutin et al. 2003). Finally, the PsbS protein found
in higher plants has four predicted MSRs (Funk et al. 1995)
and is essential for non-photochemical quenching of Chl fluorescence (Li et al. 2000). Soluble pigment–protein complexes
with photoprotective properties ranging from carotenoid-mediated quenching of Chl fluorescence to provision of a pigment–
protein environment that prevents reaction of Chl with triplet
Novel eukaryotic chlorophyll–carotenoid proteins have
evolved at least twice following the origin of the plastid and
include the widely distributed integral membrane light-harvesting complexes (LHCs) and the dinoflagellate-specific
soluble peridinin–chlorophyll proteins. In the glaucophytes, homologs of these proteins are reportedly absent.
We have identified a novel carotenoid-rich protein (CRP) in
the glaucophyte Cyanophora paradoxa that is 28 kDa and
immunologically related to the family of LHCs. CRP is
associated with the thylakoid membrane, though it can be
removed by stringent washes, suggesting that there are
probably significant structural differences between CRP
and the LHCs. CRP co-localizes with a zeaxanthin-rich thylakoid membrane fraction that also contains β-carotene,
chlorophyll and an unidentified carotenoid. Despite this, we
found no evidence for carotenoid–chlorophyll energy transfer in the isolated complex, suggesting that light harvesting
may not be a primary function. The presence of CRP in C.
paradoxa is evidence for the evolution of a novel pigmentbinding protein in the glaucophytes.
Keywords: Cyanophora paradoxa — Light-harvesting complex
— Pigment-binding protein — Plastid evolution — Zeaxanthin.
Abbreviations: CBR protein, carotenoid biosynthesis-related protein; CRP, carotenoid-rich protein; ELIP, early light-inducible protein;
FCP, fucoxanthin–chlorophyll protein; FP, free pigment; FV/FM, maximum yield of PSII; GF, green fraction; HL, high light; LHC, lightharvesting complex; LL, low light; MSR, membrane-spanning region;
PBS, phycobilisomes; PCP, peridinin–chlorophyll protein; SEP, stressenhanced proteins; TLC, thin-layer chromatography; WSC protein,
water-soluble chlorophyll protein; YL, yellow fraction.
Introduction
Photosynthetic organisms use light-harvesting antennae to
optimize the effective cross-sectional area for absorption of
light energy and to regulate transfer of excitation energy to the
photosynthetic reaction centers. In oxygenic photosynthetic
organisms, several diverse classes of proteins that perform the
function of light harvesting have evolved independently (Green
and Durnford 1996). Light-harvesting antennae are comprised
1
Corresponding author: E-mail, [email protected]; Fax, +1-506-453-3583.
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Carotenoid-rich protein in Cyanophora paradoxa
417
Fig. 1 Immunoblot of total cellular proteins from (A) the green alga
Chlamydomonas reinhardtii, (B) the red alga Rhodomela confervoides, (C) the cyanobacterium Synechococcus PCC7942 and (D) the
glaucophyte Cyanophora paradoxa. Lanes were normalized to total
cell protein per lane. Proteins were separated by SDS–PAGE and visualized by Western blot analyses with an antibody raised to the fucoxanthin–Chl protein of a marine raphidophyte (anti-FCP).
oxygen have also evolved independently. Within this group are
the soluble carotenoid proteins of cyanobacteria (Wu and Krogmann 1997) and the water-soluble chlorophyll protein (WSC)
found in higher plants (Satoh et al. 2001).
It is widely accepted that plastids of algae and plants arose
from an endosymbiotic association between a cyanobacterium
and a eukaryotic host (Delwiche 1999). Of the three separate
algal groups believed to have obtained plastids via a single primary endosymbiotic event—red algae, green algae and glaucophytes—only the red and green algae are known to posses
LHCs. The presence of LHCs in these divisions to the exclusion of cyanobacteria supported a monophyletic origin of the
primary plastids (Wolfe et al. 1994). The glaucophytes have
long been regarded as an evolutionary oddity due to the presence of a remnant peptidoglycan wall surrounding the plastid.
Though the plastid clearly evolved from a cyanobacterium,
they often form only a weak sister group to the red and green
algal lineages (Moreira et al. 2000); thus the origin of their
plastids has not been convincingly resolved. A study using two
different antisera to LHCs failed to find any immunological
evidence for their presence in the glaucophyte, Cyanophora
paradoxa (Koike et al. 2000), which suggests that LHCs either
evolved following the divergence of glaucophytes from the red
and green algal lineages, or that there was an independent plastid acquisition event.
To resolve the evolution of the eukaryotic LHCs further,
we examined the pigment–protein complexes in C. paradoxa.
We have identified a pigment-binding protein in C. paradoxa,
carotenoid-rich protein (CRP), which is immunologically
related to the LHCs but with different biochemical properties.
The potential role of CRP in C. paradoxa and its implication
for the evolution of antennae complexes will be discussed.
Fig. 2 (A) Proteins from whole cells (WC), cyanelle membranes (M)
and soluble cyanelles proteins (S) were separated by SDS–PAGE. Phycobilisomes (PBS) were visualized by enhanced fluorescence following staining with ZnSO4. (B) CRP and D1 were visualized by Western
blot analyses with anti-FCP and anti-D1 (an antibody derived from the
D1 protein of PSII). (C) Thylakoid membrane proteins (M), membranes washed with 0.5 mM NaCl (WM) and proteins removed by
washing with 0.5 mM NaCl (Su) were visualized by Western blot analyses with anti-FCP and anti-D1 antisera. (D) Cyanelle membrane proteins were separated by Triton X-114 phase partitioning into
membrane (TrM) and soluble (TrS) phases. Proteins were visualized
by Western blot analyses with anti-FCP and anti-D1. (E) Thylakoid
membranes were either untreated (Th) or extracted with chloroform :
methanol (5 : 4 v/v) and separated into soluble (S) and insoluble (In)
fractions. The presence of D1 and CRP in each of these fractions was
determined by Western blot using the anti-D1 and anti-FCP antisera
(F) Thylakoid membranes from C. reinhardtii (C.r.) and C. paradoxa
(C.p.) were incubated without (–T) or with (+T) trypsin and analyzed
by Western blot analysis with anti-FCP.
Results
C. paradoxa possesses a pigment-binding protein that is associated with thylakoid membranes and is immunologically related
to LHCs
Using an antibody (anti-FCP) derived against a fucoxanthin–chlorophyll protein from Heterosigma carterae, a marine
raphidophyte, we detected a 28 kDa protein in whole-cell
extracts that we will refer to as CRP for carotenoid-rich protein (Fig. 1A). Anti-FCP also cross-reacts with LHCs from
both red and green algae, detecting at least three apoproteins of
photosystem II (PSII) in the green alga Chlamydomonas reinhardtii (25, 30 and 33 kDa) and two LHCI apoproteins in the
red alga Rhodomela confervoides (14 and 18 kDa) (Fig. 1B,
D), while no immunologically related bands were detected in
the cyanobacterium Synechococcus PCC7942 (Fig. 1C).
To determine the subcellular localization of CRP,
cyanelles were isolated and separated into soluble and mem-
418
Carotenoid-rich protein in Cyanophora paradoxa
Fig. 3 (A) Thylakoid membranes of C. paradoxa
were treated with 0.1% β-D-maltopyranoside and
fractionated by sucrose density centrifugation,
resulting in three fractions (depicted diagrammatically): a minor, free pigment (FP) fraction, an upper
yellow fraction (YF) and a lower green fraction
(GF). (B) Proteins from whole (Th) and fractionated
thylakoid membranes were separated by SDS–PAGE
and visualized by Western blot analyses with antiFCP and anti-D1 antisera. (C and D) Absorbance
spectra in 90% methanol are shown for the upper YF
(C) and for the lower green fraction (D). The absorbance spectrum of thylakoid membranes is shown as a
reference.
brane fractions. The efficiency of separation was verified by
analyses of pigment and protein contents of the two fractions.
The cyanelle membrane fraction contained all of the Chl a and
carotenoids and only trace amounts of PBS chromophores relative to the soluble fraction (Fig. 2A). Immunoblot analyses of
membrane and soluble fractions with anti-FCP and anti-D1
showed that CRP is localized to the thylakoid membrane fraction of cyanelles (Fig. 2B). Unexpectedly, CRP appears to be
only tightly associated with thylakoid membranes as it is partially removed by high-salt washes (Fig. 2C) unlike the LHCs
of Chlamydomonas that remained in the membrane fraction
(data not shown). To explore the properties of the CRP further,
we used the technique of phase partitioning with Triton X-114,
which separates integral and peripheral membrane proteins
(Bordier 1981). Following phase partitioning, CRP was
retained predominantly in the soluble phase, supporting the
observation that it is easily dissociated from the membrane
(Fig. 2D). D1, a known integral membrane protein, was present
in the soluble and membrane phase, a phenomenon that has
been reported previously for thylakoid membrane proteins (Zak
et al. 1999) (Fig. 2D). Cyanelle membranes were also extracted
with chloroform : methanol (5 : 4, v/v), a procedure that is able
to solubilize hydrophobic membrane proteins. When cyanelle
membrane proteins were extracted with this organic solvent,
CRP appeared in the insoluble fraction while D1 was found in
the chloroform : methanol-soluble phase (Fig. 2E). This indicated that CRP is not very hydrophobic and if it is a membrane
protein there is probably a large soluble portion. When cyanelle
membranes were treated with trypsin, the CRP size was unaltered, in contrast to the LHCs of Chlamydomonas that were
partially digested (Fig. 2F). CRP remained intact following
20 min of trypsin treatment (data not shown), suggesting that
its association with the membrane affords protection against
proteolytic damage.
CRP is associated predominantly with zeaxanthin
To characterize further the biochemical properties of CRP,
cyanelle membranes from C. paradoxa were fractionated by
sucrose density centrifugation following β-D-maltopyransoside
solubilization, resulting in two predominant pigmented fractions: an upper yellow fraction (YF) and a lower green fraction
(GF) (Fig. 3A). Proteins from both fractions were analyzed by
SDS–PAGE, and Western blot analyses demonstrated the presence of the PSII reaction center protein D1 in the GF (Fig. 3B).
Though there were traces of the CRP in the GF, it was very
abundant in the YF (Fig. 3B) and significantly enriched on a
per protein basis relative to thylakoid membranes. D1 was
absent in the YF, suggesting that very little cross-contamination
occurred between the two fractions.
The absorbance spectrum of the GF was similar to that of
whole thylakoid membranes (Fig. 3C). In contrast, the YF was
rich in carotenoids and contained a smaller amount of Chl (Fig.
Fig. 4 Absorption spectra of the three pigments isolated following
TLC separation of the YF from the sucrose gradient. The orange-yellow band (λmax 452, 479) and orange bands (λmax 450) were dissolved
in 95% ethanol, while the green band was eluted in 80% acetone (λmax
415, 663). The orange-yellow band on the TLC plate was yellow when
eluted in 95% ethanol.
Carotenoid-rich protein in Cyanophora paradoxa
Fig. 5 Yellow fractions (YFs) obtained from sucrose density centrifugation of C. paradoxa thylakoid membranes (Th) were separated further by anion exchange chromatography. (A) Absorbance spectra of
the flow-through (F/T, solid line) and the pigmented band (Ax, dashed
line) obtained following a 0.5 M NaCl wash are shown. (B and C) Proteins were separated by SDS–PAGE and visualized by Western blotting with anti-FCP (B) and silver staining (C). CRP was not detected
in the F/T fraction (data not shown).
3D). The molar ratios of carotenoids to Chl in the GF and YF
were 0.23 ± 0.04 and 2.3 ± 0.6, respectively. The predominant
carotenoids that accumulate in C. paradoxa are zeaxanthin, βcarotene (Chapman 1966) and β-cryptoxanthin in small quantities (Schmidt et al. 1979), and pigments in the YF were separated by thin-layer chromatography (TLC) to determine the
proportion of each (Fig. 4). The major pigment component of
the YF was identified as zeaxanthin (orange-yellow band, rf =
0.59) based on previous studies (Chapman 1966, Schmidt et al.
1979) and its absorption maxima in ethanol (Davies and Köst
1988). Chl a (green band, rf = 0.73) and β-carotene (orange
band, rf = 0.98) were also present in this fraction (Fig. 4). A
thin orange band running near the front of the Chl a band was
visible on the TLC plate. This pigment may be β-cryptoxanthin as discussed by Schmidt et al. (1979). The presence of a
419
Fig. 6 (A) Thylakoid membranes from C. paradoxa were solubilized with 1% (w/v) β-D-maltopyranoside and separated by non-denaturing gel electrophoresis on an 8% (w/v) polyacrylamide gel.
Pigmented protein bands (P1–P3) and the free pigment band (FP) are
indicated. Gels were exposed to UV light in order to visualize fluorescent bands (right panel). Pigmented bands were excised from nondenaturing polyacrylamide gels and proteins were eluted. (B) Proteins
were separated by SDS–PAGE and visualized by silver staining. (C)
Western blot analyses with anti-FCP. An abundant 28 kDa protein
detected by silver staining in P3 may correspond to the protein exhibiting immunoreactivity with anti-FCP (CRP).
carotenoid is obvious in the spectra of the green (Chl a) band
of the YF, which has the unexpected shoulder from 450 to
500 nm (Fig. 4). This carotenoid was not detected in the Chl
band from the GF, suggesting that it is exclusively associated
with the CRP. Though there was variability in the pigment content of the YF, probably due to the labile nature of the complex,
the molar ratios of the pigments on average were: 1 Chl a; 0.2
± 0.02 unknown carotenoid (‘β-cryptoxanthin’); 0.5 ± 0.4 βcarotene; 3.2 ± 1.1 zeaxanthin. The free pigment (FP) fraction
at the top of the gradient was composed almost entirely of βcarotene with a trace amount of Chl a only visible under UV
light (data not shown). When proteins from the YF were purified further by anion exchange chromatography, a pigmented
band containing both Chls and carotenoids was eluted from the
420
Carotenoid-rich protein in Cyanophora paradoxa
Fig. 8 Analysis of total cellular proteins extracted from cultures of C.
paradoxa acclimated to LL or HL for 24 h. Proteins from an equivalent number of cells were separated by SDS–PAGE. (A) Phycobilisomes (PBS) were visualized by ZnSO4 staining. (B) CRP was
visualized by Western blot analysis with anti-FCP. (C) Changes in D1
were visualized using the anti-D1 antiserum.
Fig. 7 Room temperature fluorescence characteristics of the yellow
fraction (top panel) and thylakoid membranes (bottom panel). The
excitation spectrum (em680, emission at 680 nm) and emission spectra excited at either 436 nm (ex436) or 487 nm (ex487) were determined for the YF from both the sucrose gradient (top panel) and
thylakoid membranes (bottom panel). The absorbance spectra for each
are provided (dashed lines). The absorbance spectrum of the soluble
phycobilisome fraction (PBS Abs) is also provided for comparison in
the bottom panel.
column following a wash with 0.5 M NaCl (Fig. 5A). Western
blot analyses verified that the pigmented band was enriched in
CRP, further verifying the association of this protein with pigments (Fig. 5B). Free pigment consisting primarily of carotenoid was eluted from the column with salt-free wash buffer (Fig.
5A) indicative of the labile nature of this pigment–protein.
A similar carotenoid-rich fraction was detected by an
independent biochemical technique that fractionated detergentsolubilized thylakoid membranes by non-denaturing gel electrophoresis. Three pigmented bands were resolved in C. paradoxa thylakoid membranes (P1–P3) in addition to a FP band
comprised predominantly of carotenoids (Fig. 6A). As expected,
the higher molecular weight bands containing photosynthetic
reaction centers lacked auto-fluorescence upon exposure to UV
light, in contrast to P3 (Fig. 6A). SDS–PAGE analyses of proteins eluted from band P3 indicated it was enriched in a 28 kDa
protein that was absent in P1 and P2 (Fig. 6B). Western blot
analyses of proteins eluted from P2 showed an enrichment of
D1, suggesting that P2 corresponds to PSII complexes. P3 was
enriched in CRP (Fig. 6C).
Though the YF absorbs light strongly in the 450–520 nm
region due to the abundance of carotenoids, there was little evidence for efficient transfer of excitation energy directly to chlorophyll when the complex was excited at 487 nm, compared
with 436 nm where Chl absorbs strongly (Fig. 7, top panel).
The small amount of emission at 680 nm when excited at
487 nm appears to be residual Chl absorption, as boiling the
sample led to similar, small declines in fluorescence yield at
both excitation wavelengths (data not shown). Emission at
680 nm was significant only when Chl was directly excited
(<450 nm) (Fig. 7). Since this complex was purified with detergents, energy transfer could have been disrupted, therefore we
conducted similar measurements on purified thylakoid membranes (Fig. 7, lower panel). In thylakoids, excitation at 487 nm
still did not yield evidence for efficient energy transfer from
carotenoid to Chl, despite the abundance of carotenoids in this
fraction. As with the YF, Chl emission at 680 nm was generated primarily by wavelengths where Chl absorbs maximally.
The increase in fluorescence emission in thylakoids when
excited at >510 nm is due to the absorption of PBS that
remained attached during purification of thylakoid membranes.
CRP accumulation is down-regulated in response to high light
To determine whether the co-localization of CRP with the
carotenoid-rich fraction of thylakoid membranes may be indicative a photoprotective function, we investigated the regulation of CRP accumulation in response to high light (HL).
Photoacclimation was initiated by a 10-fold increase in growth
irradiance, and changes in CRP accumulation in C. paradoxa
were monitored after 24 h. The maximum yield of PSII (FV/FM)
was reduced by 20% in HL-grown cultures, indicating that this
Carotenoid-rich protein in Cyanophora paradoxa
421
Table 1 Pigment content and maximum yield of PSII (FV/FM) in LL- and HL-acclimated C. paradoxa
cultures
LL
HL
pg of Chl a cell–1
pg of carotenoid cell–1
FV/FM
Max O2 evolution (fmol cell–1 h–1)
0.202 ± 0.014
0.241 ± 0.012
0.049 ± 0.003
0.076 ± 0.004
0.589 ± 0.004
0.467 ± 0.028
18.6
11.1
Saturation of oxygen evolution rates occurred at photon flux densities (PFDs) of 100 and 300 µmol photons m–2 s–1
for LL- and HL-acclimated cultures, respectively.
was a significant light stress (Table 1). Following a 24 h exposure to HL, Chl a content per cell increased slightly although
the amount of Chl per protein in thylakoid membranes was
reduced by 14% (Table 1). The differences in pigment composition on a per cell basis can be attributed to changes in the
density of thylakoid membrane components in HL-acclimated
cells, rather than an increase in concentration of Chl-binding
proteins in the thylakoid membranes. Total carotenoid content
per cell, however, increased 50% following a shift into HL
(Table 1). Fractionation of thylakoid membranes revealed a 5fold increase in the amount of FP isolated from HL-exposed
cultures (Table 1) that is comprised primarily of β-carotene as
determined by TLC (data not shown). Therefore, the increase
in carotenoids in HL-acclimated C. paradoxa is likely to be
accounted for by an increase in membrane-localized β-carotene, as opposed to carotenoids associated with pigment–protein complexes, such as the CRP.
Photosynthetic irradiance response curves for low light
(LL)- and HL-acclimated cultures show a light saturation point
for O2 evolution of 100 and 300 µmol photons m–2 s–1, respectively (Table 1). The requirement for a higher light intensity to
saturate photosynthesis in HL-acclimated cultures suggests a
down-regulation in light-harvesting antennae size, a phenomenon that is a common response of photosynthetic organisms to
increases in irradiance levels (Anderson et al. 1995). In support of this reduced light-harvesting capacity, there was a
decrease in PBS fluorescence following HL exposure (Fig. 8A)
that was paralleled by a reduction in CRP content during this
same period (Fig. 8B). The concentration of PSII reaction centers per cell also declined following HL exposure (Fig. 8C).
Discussion
Identification and characterization of a novel pigment-binding
protein in C. paradoxa
Using an antibody derived against an FCP, we were able
to detect a thylakoid-localized protein in C. paradoxa that is
immunologically related to LHCs. The labile nature of its association with the thylakoid membrane (Fig. 2), however, is quite
unusual given its immunological relatedness to a pigment-binding protein with three MSRs. Different extraction procedures
on cyanelle membranes suggest that this membrane protein is
not very hydrophobic and is easily liberated from its association with the thylakoid membrane. The location of CRP in the
thylakoid membrane offers it protection from trypsin digests,
indicating that any loop or soluble regions are not accessible
due to its association with the membrane or other proteins.
Though the cross-reactivity of the CRP with the FCP
antisera indicates the presence of shared epitopes, there are
likely to be significant structural differences between it and the
LHC gene family. First, although CRP cross-reacts with the
FCP antisera, it only cross-reacts very weakly with a
Chlamydomonas LHC-specific antiserum (data not shown), or
not at all, as was experienced by another group (Koike et al.
2000). This indicates that the CRP and the LHCs share relatively few antigenic epitopes, and the immunoreactivity to the
FCP antiserum could be explained by the presence of pigmentbinding motifs that are specific to the FCPs. Second, the CRP
can be partially dissociated from thylakoid membranes, suggesting that it is not as hydrophobic as the LHC protein family.
Although the LHC-related, LI818 protein of C. reinhardtii displays similar biochemical properties in that it can be partially
removed by salt washes and alkaline treatments (Richard et al.
2000), we are not certain that CRP is a bona fide LHC. It is
equally possible that a novel pigment-binding protein has
evolved from small LHC-like proteins such as the HLIPs,
which are present in Cyanophora. It is also conceivable that the
CRP is not an LHC-related protein but that it only shares structural motifs that participate in pigment binding, that may
account for the immunological similarity. One example of this
is found in the WSC proteins, which share the [F/Y]DPLGL
motif that is conserved in LHCs (Satoh et al. 2001). We are
working towards sequencing the protein and cloning the gene
to determine the basis of this cross-reactivity.
Several biochemical techniques were used to examine the
association of CRP with photosynthetic pigments. Separation
of thylakoid membrane proteins by sucrose gradient fractionation, anion exchange chromatography and non-denaturing gel
electrophoresis resulted in isolation of a CRP-enriched fraction
containing zeaxanthin, chlorophyll, β-carotene and a small
amount of another carotenoid (Fig. 3–5), tentatively identified
as β-cryptoxanthin based on the work of Schmidt et al. (1979).
Under these mild, non-denaturing conditions and during anion
exchange chromatography, the bound zeaxanthin is easily dissociated, suggesting that pigments are non-covalently bound to
CRP (Fig. 5A). The correlation of the immunoreactive 28 kDa
band with a carotenoid-rich fraction in three independent fractionation procedures indicates that the co-migration of a non-
422
Carotenoid-rich protein in Cyanophora paradoxa
Table 2 Pigment content of membranes and free pigments
from sucrose density-fractionated membranes of cyanelles isolated from LL- or HL-acclimated C. paradoxa
LL
Membranes
Free pigment
HL
Membranes
Free pigment
ng of Chl a
ng of carotenoids
65.7 ± 1.1
t
13.2 ± 0.8
2.5 ± 0.4
56.6 ± 4.9
t
13.2 ± 1.0
13.4 ± 0.7
Pigments are expressed as ng µg–1 of protein for membrane fractions
and ng µl–1 of sample for free pigment fractions. There were only trace
(t) amounts of Chl in the free pigment fractions.
specific band that cross-reacts with this antiserum is unlikely
and that this protein actually shares epitopes with the FCP family of proteins.
While the pigment-binding properties of LHCs are quite
diverse, the molar ratio of carotenoids to Chls typically ranges
from 0.5 to 1.3 in LHCs found in chlorophytes, rhodophytes
and chromophytes (Hiller et al. 1993, Bassi and Caffarri 2000,
De Martino et al. 2000, Grabowski et al. 2001). CRP, however,
has a relatively high carotenoid to Chl ratio, which is often
indicative of a photoprotective role rather than light harvesting, as is the case with the ELIPs of higher plants (Adamska et
al. 1999) and the carotenoid biosynthesis-related (CBR) protein of Dunaliella salina (Banet et al. 2000). In all cases, exposure to HL stress leads to an induction of these proteins
(Havaux et al. 2003). In contrast, LHCs of algae and higher
plants that are involved in light harvesting are down-regulated
in response to increased irradiance, as a smaller antennae size
is necessary to absorb an efficient number of photons to drive
photochemistry without over-excitation of the reaction centers
(Anderson et al. 1995). During HL acclimation in C. paradoxa, the total carotenoid content increased on a per cell basis
(Table 1), although the total amount of carotenoids localized to
thylakoid membranes remained constant following HL acclimation (Table 2). An increase in ‘free carotenoids’ in HL-acclimated C. paradoxa is not surprising, given that such
carotenoids have roles in preventing peroxidation of plastid
membranes (Havaux 1998) and in the regulation of membrane
fluidity (Ourisson and Nakatani 1995). Despite the increase in
total cellular carotenoids, CRP accumulation was down-regulated in concert with PBS following HL acclimation. Although
the decrease in CRP would suggest a role in light harvesting,
we were unable to detect significant carotenoid to Chl energy
transfer in the YF from the sucrose gradient or in thylakoid
membranes. If not an antenna, then CRP is likely to participate
in photoprotection or the regulation of energy distribution to
the reaction centers by an unknown mechanism. Given the
abundance of zeaxanthin, a role in energy dissipation is a
strong possibility. The decline in HL indicates that CRP is not
strictly associated with stress like the ELIPs and CBR protein
of Dunaliella, the latter of which is also proposed to bind zeaxanthin (Levy et al. 1993). Instead, CRP is abundant under our
LL conditions and thus has an important role in the
‘unstressed’ state. Although a decline in CRP abundance in HL
seems to be counter to a proposed role in photoprotection, it
may function in fixed stoichiometry with respect to reaction
centers or PBS, which both decline under HL.
CRP may represent a unique innovation in the regulation
of light harvesting, although it is unclear at this stage whether
CRP is involved in energy dissipation, regulating the transfer
of excitation energy to reaction centers or if it has another role
related to photoprotection or light harvesting. Further studies
are necessary to understand the pigment-binding properties of
CRP and its role in C. paradoxa. Elucidating the nature of light
harvesting and photoprotective strategies employed by this
enigmatic photosynthetic eukaryote will provide insight into
the evolution and diversification of antennae systems that followed the acquisition of plastids.
Materials and Methods
Growth conditions and collection of algae
Cyanophora paradoxa Korsh strain LB555 obtained from the
Culture Collection of Algae (University of Texas at Austin) were
grown in modified WCg medium buffered with 10 mM HEPES-NaOH
(pH 7.5) bubbled with humidified air (Guillard and Ryther 1962, Guillard 1975). C. paradoxa cultures were grown to a maximum cell density of 1×106 cells ml–1 at 24°C in LL (55 µmol photons m–2 s–1). To
induce a light stress, cultures were shifted to HL (500 µmol photons
m–2 s–1) for 24 h. C. reinhardtii strain CC125 was grown in Tris-acetate-phosphate medium as described (Durnford et al. 2003). R. confervoides (Rhodophyta; Subclass Florideophycidae) was collected at
Green’s Point (Letete, NB, Canada). Synechococcus PCC7942 was
obtained from Dr. D. Campbell.
Protein extraction from whole cells and isolated cyanelles
For extraction of total cellular proteins from C. paradoxa, C.
reinhardtii and Synechococcus PCC7942, 5×107 cells were harvested
and proteins extracted in 50 µl of lysis solution containing 0.2 M
Na2CO3, 2% SDS (w/v), 5 mM aminocaproic acid and 1 mM benzamidine HCl. R. confervoides tissue (500 mg) was frozen in liquid N2 and
ground to a fine powder with a mortar and pestle and resuspended in
500 µl of lysis solution. The mixture was incubated at 60°C for
10 min. Following lysis, all samples were centrifuged at 14,000×g for
5 min and the resulting supernatant was used for protein analyses.
Cyanelles were isolated from C. paradoxa (∼7×109 cells) and
lysed as previously described (Koike et al. 2000). Lysed cyanelles
were centrifuged at 150,000×g for 1 h at 4°C. The soluble blue supernatant was removed and used directly for further analyses. The pellet,
containing crude thylakoid membranes, was resuspended in HEMS
buffer [50 mM HEPES-NaOH (pH 7.5), 2 mM EGTA, 1 mM MgCl2
and 0.5 M sucrose] to a final protein concentration of 5 µg µl–1.
Thylakoid membrane treatments
To examine association of proteins with membranes, crude thylakoid membranes were prepared as described above and washed with
0.5 M NaCl in HEMS buffer for 20 min at 4°C followed by centrifuga-
Carotenoid-rich protein in Cyanophora paradoxa
tion at 150,000×g for 30 min. Supernatants were collected and concentrated by precipitation in 90% (w/v) acetone and membranes were
resuspended in HEMS buffer. Protein concentrations were determined
using the BCA protein assay (Pierce; Rockford, IL, U.S.A.). Cyanelle
membranes were subjected to Triton X-114 phase partitioning as previously described (Bordier 1981) and also extracted in chloroform :
methanol [5 : 4 (v/v)] according to Ferro et al. (2000).
Thylakoid membrane fractionation and anion exchange chromatography
Crude thylakoid membranes isolated from C. paradoxa were
resuspended in HEMS buffer to a final Chl concentration of 0.5 (mg
Chl) ml–1 and solubilized with 1% β-D-maltopyransoside (w/v) for
10 min at 4°C. The suspension was centrifuged at 8,600×g for 5 min at
4°C, and the supernatant placed on a linear sucrose gradient [5-30%
(w/v) sucrose in HEMS buffer] and centrifuged at 150,000×g for 14 h
at 4°C. Sucrose gradients were created using the Gradient Master (BioComp Inst. NB, Canada). Colored fractions were removed from the
gradients and concentrated using a 5 kDa cut-off centrifugal filter unit
prior to further analyses (Millipore; Billerica, MA, U.S.A.).
Concentrated sucrose gradient fractions were loaded onto a column containing DEAE anion exchange resin (Amersham Pharmacia;
Uppsala, Sweden) pre-equilibrated with 50 mM HEPES and 0.01% βD-maltopyransoside (w/v). Washes were performed with 10 and
500 mM NaCl. Proteins were precipitated from collected wash fractions with 100% (w/v) acetone and resuspended in HEMS buffer.
Non-denaturing gel electrophoresis
Following detergent solubilization of thylakoid membranes, as
described above, samples containing 15 µg of Chl were separated
under non-denaturing conditions on 8% (w/v) polyacrylamide gels
(Allen and Staehelin 1991). Pigmented bands were excised from the
gel and proteins were eluted by incubation at 90°C for 15 min in
sample loading buffer [125 mM Tris–HCl, pH 6.8; 2% (w/v) SDS; 2%
(w/v) β-mercaptoethanol; 0.05% (w/v) bromophenol blue, 20% (v/v)
glycerol].
SDS–PAGE and immunoblot analyses
Protein samples were separated by electrophoresis on 14% Tris–
glycine, SDS–polyacrylamide gels (Sambrook et al. 1989). For comparisons of protein levels between light regimes, samples were loaded
on an equal cell basis (equivalent to protein extracted from 1.9×106
cells). For cross-species comparisons and all other analyses, proteins
were loaded on an equal protein basis (20 µg). To compare relative
levels of PBS, gels were stained with 75 mM ZnSO4 (Raps 1990). For
detection of proteins following SDS–PAGE, gels were subjected to silver staining as previously described (Oakley et al. 1980). Immunoblot
analyses were done as previously described (Durnford et al. 2003).
Antiserum directed against the core reaction center of PSII (D1) was
provided by Dr. D. Campbell. The anti-FCP antiserum was made
against the FCPs of a marine chromophyte (Heretosigma carterae;
Harnett 1998) and was provided by Dr. B.R. Green.
Pigment extraction, thin layer chromatography and quantification
Chls and carotenoids were extracted from cell pellets and the
pigment concentrations were determined using previously published
molar extinction coefficients (Lichtenthaler 1987, Tandeau de Marsac
and Houmard 1988). Absorbance spectra were measured with a Cary
100 spectrophotometer (Varian, Palo Alto, CA, U.S.A.). Sucrose gradient fractions were extracted in 100% acetone followed by petroleum
ether. The petroleum ether layer containing the pigments was removed
and the pigments concentrated by evaporation under N2 gas and spotted on a silica gel 60 TLC plate (0.25 mm thickness). Pigments were
423
separated with petroleum ether : isopropanol : water mixture (100 :
10 : 0.25, by vol.) in the dark. Bands containing pigments were
scraped off the plate and eluted in 95% ethanol (carotenoids) or 80%
acetone (Chl) and quantified as described above.
Oxygen evolution and fluorescence measurements
Cyanophora paradoxa cells were concentrated to a cell density
of 8×106 cells ml–1 for oxygen evolution and fluorescence measurements. Oxygen evolution and the maximum efficiency of PSII (FV/FM)
were measured as described previously (Durnford et al. 2003). Room
temperature fluorescence and excitation measurements were conducted on a Perkin-Elmer LS50 spectrophotometer equipped with a
red-sensitive photomultiplier. Samples were diluted in HEMS to a concentration of approximately 1–2 µg ml–1 prior to measuring.
Acknowledgments
We are grateful to Drs. B.R. Green, D. Campbell and K. Hoober
for providing the FCP, D1, and Chlamydomonas LHC antisera, respectively. We thank Dr. G. Saunders for assisting with the collection of
Rhodomela confervoides, and Dr. V. Erickson and I. Erickson for
assistance in translating German. This work was supported by a grant
from the National Sciences and Engineering Council of Canada. H.R.
is funded under the auspices of PEP, the Protist EST Program, which is
funded by Genome Canada, Genome Atlantic and the Atlantic Innovation Fund.
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(Received March 23, 2004; Accepted December 10, 2004)