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INTRODUCTION |
Xanthophylls are a class of carotenoids associated with the light
harvesting complexes of plant chloroplast membranes (1). In most
plants, there are three major xanthophylls, namely lutein, neoxanthin,
and violaxanthin, the last of which can be reversibly de-epoxidized to
antheraxanthin and zeaxanthin via the xanthophyll cycle (2). The reason
for this diversity in xanthophyll composition is not entirely clear,
although the conservation of xanthophyll composition across a range of
plant species (3-5) indicates a specific role for each one. Although
xanthophylls are bound to both
LHCII1 and LHCI, the nature
of the binding has not been determined, and there are significant
differences in the values reported for the numbers of pigments bound to
particular complexes (6-11). In the structural model for the major
LHCII component, LHCIIb, there are two carotenoid molecules that are
presumed to be the two luteins that have been shown to be bound by this
complex (12). No other carotenoids were detected in this
crystallographic study, despite the fact that there are either one or
two other carotenoids present. For the other LHCII components, CP29,
CP26, and CP24, there is even less certainty, with estimates of the
number of bound carotenoids differing significantly (see reviews in
Refs. 9 and 11).
In the case of the xanthophyll cycle carotenoids, establishing the
stoichiometry of binding is of particular importance because this cycle
plays a major role in controlling the efficiency of light harvesting
(5, 13, 14): in light-limiting conditions, maximum efficiency of light
harvesting is associated with the presence of violaxanthin, whereas
de-epoxidation to zeaxanthin is correlated with down-regulation of
light harvesting efficiency because of the nonradiative dissipation of
excess energy. This latter process is important in the protection of
the photosynthetic system from photodamage and is readily detected as
the nonphotochemical quenching of chlorophyll fluorescence. For LHCIIb,
the reported values for xanthophyll cycle carotenoid binding range form
trace amounts (7) up to 1 per trimer (6). For CP29 and CP26, there are
reports of between 0.65 (7, 9) and 1.5 (6) xanthophyll cycle
carotenoids bound. This range of values gives rise to very different
conclusions about the location of the xanthophyll cycle and its mode of
action. With only trace amounts of xanthophyll cycle in LHCIIb,
attention is focused on the minor complexes as the site of
nonphotochemical quenching. The lowest estimates of the content of
xanthophyll cycle carotenoids bound to these complexes lead to the
suggestion that one of the internal luteins in the LHCIIb model is
replaced by a violaxanthin (9). This internal location is consistent
with a view that zeaxanthin, formed by de-epoxidation of this
violaxanthin, directly quenches Chl excited states via Chl-car energy
transfer (15, 16). Conversely, if one violaxanthin is bound per LHCIIb
trimer, then at least 50% of the violaxanthin pool is associated with
the main population of LHCII, bound at a site other than the lutein
sites (8). Similarly, higher stoichiometries of binding to the
minor complexes indicate that at least a proportion of the violaxanthin
pool is not bound to the internal "lutein sites." Such data are
consistent with a peripheral location of violaxanthin and with the
hypothesis that the xanthophylls cycle controls the structure of the
LHCII system (13, 17, 18). Central to these arguments is also the
question of which violaxanthin molecules can be de-epoxidized to
zeaxanthin. In fact, the maximum de-epoxidation state of CP29 is rather
low compared with LHCIIb (8), suggesting that peripheral violaxanthin
is the preferred substrate for the violaxanthin de-epoxidase. A further
question is whether violaxanthin and zeaxanthin are bound with equal
strengths to LHCII; it has been suggested that only zeaxanthin (and
antheraxanthin) can be associated with the complexes so as to initiate
nonphotochemical quenching (14).
In order to understand the way in which the xanthophyll cycle
carotenoids, and the other xanthophylls, control the structure and
function of LHCII, it is necessary to determine where and in what
numbers these carotenoids are associated within the LHCII system. In
this paper, we describe a systematic study of the binding stoichiometries of pigments to both the minor and major LHCII components and determine the relative binding strengths.
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EXPERIMENTAL PROCEDURES |
Thylakoids and photosystem II BBY particles were prepared from
spinach leaves as described previously (8). Light harvesting complexes
of photosystem II were prepared from spinach by isoelectric focusing of
either BBY particles or unstacked isolated thylakoids. The thylakoids
were suspended in 0.33 M sorbitol, 1 mM EDTA,
50 mM HEPES, pH 7.6, and solubilized in 20 mM
n-dodecyl
-D-maltoside (DM). To induce de-epoxidation of
the xanthophyll cycle, spinach leaves were illuminated prior to
isolation of BBY particles as described previously (8). Alternatively,
in order to achieve the maximum de-epoxidation state, unstacked
thylakoids were incubated in 0.33 M sorbitol, 1 mM EDTA, 30 mM HEPES, 20 mM MES, 40 mM ascorbate at pH 5.5 at 20 °C for 30 min (19). After
incubation, thylakoids were diluted, centrifuged, and resuspended ready
for solubilization and IEF, which was performed as described previously
(8). Further purification of LHCII was carried out by sucrose density
gradient centrifugation: sucrose gradients were seven step exponential gradients from 0.15 to 1.0 M sucrose dissolved in 20 mM HEPES buffer containing 20 µM DM. The run
time was 18 h at 200,000 × g in a SW41 rotor at
4 °C. To remove pigments from LHCII, samples were incubated in
different concentrations of detergent prior to centrifugation.
Fractionation of thylakoids involved treatment of unstacked thylakoids
with 10 mM DM with a ratio of DM/Chl of 8 on ice for 45 min, followed by centrifugation through a sucrose gradient. For
fractionation of BBY particles the same procedure was used except that
DM concentration was 8 mM (DM/Chl = 3).
The pigment composition of thylakoids, BBY particles, thylakoid
subfractions, and LHCII samples was determined by reverse phase HPLC
(4). SDS-polyacrylamide gel electrophoresis was used to determine the
polypeptide composition of thylakoid membrane fractions. Absorption
spectra were recorded using an Aminco DW2000 spectrophotometer, and
fluorescence emission spectra at 77 K were measured as described
previously (20, 21).
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RESULTS |
Carotenoid Content of LHCII--
Table
I presents the carotenoid content of
LHCIIb, CP29, and CP26 isolated by IEF of PSII membranes. Based on the
known number of Chl b molecules bound to each complex (9) and the
measured Chl a/b ratios, it was possible to determine the
number of carotenoids bound. For LHCIIb, it was estimated that there
were 2 molecules of lutein, 1 of neoxanthin, and a substoichiometric
amount of violaxanthin (0.2 per monomer). CP26 bound over 1 molecule of lutein and 1 molecule each of neoxanthin and violaxanthin, whereas CP29
bound approximately 1 molecule of lutein, just over 1 of violaxanthin,
and 0.6 of neoxanthin. Using these values, and assuming that a PSII
unit in the thylakoid membrane contains 1 molecule each of CP29, CP26,
and CP24 and 5 trimers of LHCIIb, an estimate of the carotenoid
composition of PSII can be made. These data predict that there is a
maximum of 8 molecules of violaxanthin bound to PSII and that
violaxanthin accounts for up to 10% of the total carotenoid pool.
However, assay of the carotenoid content of the BBY PSII membranes used
for the isolation of the complexes indicates that the content of
violaxanthin was almost 20% of total carotenoid, a value consistent
with measurements made for whole thylakoids. Thus, at least half of the
xanthophyll cycle pool associated with PSII cannot be accounted for by
the amounts bound to the purified complexes.
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Table I
Pigment composition of LHCII, thylakoid membranes, and photosystem II
BBY particles
LHCIIb, CP29, and CP26 were prepared by IEF of BBY PSII particles. PSII
(est) refers to that predicted from the composition of individual
complexes. %, carotenoid content as a percentage of total; MR,
estimated molar ratio; lut, lutein; neo, neoxanthin; vio, violaxanthin;
ant, antheraxanthin; XC, vio + ant + zeaxanthin; -car,
-carotene; car/Chl, molar ratio of total carotenoid to total
chlorophyll. Data are the averages ± S.E. of at least three
replicate assays of two or three separate preparations. For the
estimate of PSII pigment content, data on CP47/43 (37), D1/D2 (24), and
CP24 (7) were used, assuming that one PSII consisted of 1 D1/D2, 1 CP47, 1 CP43, 1 CP29, 1 CP26, 1 CP24, and 5 trimers of LHCIIb. The
chlorophyll contents of LHCII used the data in Ref 9. For BBY
particles, the molar ratios were estimated so as to give the measured
percentage composition.
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There are two explanations for this discrepancy: first, there could be
a pool of violaxanthin that is not bound to LHCII but is either free in
the lipid phase of the membrane or bound to another protein complex.
Second, violaxanthin is only rather loosely bound to LHCII and is
removed during solubilization of the BBY particles and complex
purification. To distinguish between these hypotheses, the
distribution of pigment in LHCII fractions and free pigment was
determined using sucrose gradient centrifugation following
solubilization of BBY PSII membranes with three different concentrations of detergent (Fig. 1). In
all cases, the distribution of Chl was approximately the same: about
60% in the LHCIIb trimeric band, approximately 15% in the monomer
band (mainly minor complexes CP26, CP29, and CP24), and the remaining
25% in PSII core complexes. Less than 5% of the Chl was found in the
free pigment zone. However, in contrast to these results, the
distribution of violaxanthin was very dependent on detergent
concentration. At the lowest detergent concentration, nearly 80% of
the violaxanthin was in the trimeric LHCIIb fraction, and approximately
5% was in the free pigment zone. At the highest detergent
concentration, this distribution was reversed, with over 80% in the
free pigment zone and less than 5% in the LHCIIb trimers. The
distribution of violaxanthin in the monomer fraction was approximately
20% at each detergent concentration.

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Fig. 1.
The distribution of pigment between PSII
cores, LHCII trimers, LHCII monomers, and the free pigment zone
following detergent solubilization of photosystem II BBY
particles. Inset, diagrammatic representation of
distribution of Chl in the centrifuge tube. Shown are Chl and
violaxanthin (Vio) distribution following solubilization
with 5 (8 mM), 8 (16 mM), and 14 (22 mM) molar ratio of DM to chlorophyll. Fp, free
pigment zone; mon, monomeric LHCII; tr, trimeric
LHCII; PSII, core complexes of PSII. Data are expressed as
percentage of pigment loaded onto the gradient.
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Carotenoid Composition of Oligomeric LHCII--
These data showed
that the xanthophyll cycle carotenoid not associated with purified
LHCII was loosely associated with the complexes in the thylakoid
membrane. Moreover, with low detergent concentrations, violaxanthin
could be retained bound to solubilized LHCII. Therefore, a different
approach was taken to investigating the xanthophyll cycle content of
the LHCII antenna: rather than isolating individual antenna complexes,
gentle detergent treatment was used to try to remove an intact
oligomeric PSII light harvesting system containing the complete
xanthophyll cycle pool. Treatment of either BBY particles or thylakoids
with carefully established detergent concentrations was found to
produce a series of Chl-containing bands on a sucrose gradient (Fig.
2). For BBY particles, there was less
than 3% free Chl arising from this treatment, whereas there was none
detectable with thylakoids. At 0.73 M sucrose, the band
that contained 20-30% of the total Chl was referred to as the
A-band.

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Fig. 2.
Sucrose density gradient profiles of
thylakoids and photosystem II BBY particles after solubilization of
thylakoids (1) in 10 mM DM and BBY
particles (2) in 8 mM DM.
The antenna (A) band recovered from the first gradient was
solubilized in either 6 mM DM (3) or 0.2 mM DM (4) and recentrifuged. On the
left of each tube is the sucrose molarity. On the
right of each tube is the amount of Chl in each fraction
expressed as a percentage of the total applied.
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The A-band had a Chl a/b ratio of 1.61 and a low content of
-carotene compared with BBY particles, suggesting a deficiency in
the CP47/CP43/RC core (Table I). The carotenoid/Chl ratio was higher
than either purified LHCII or PSII, and the violaxanthin content was
26% of total carotenoid. The absorption spectrum was very similar to a
spectrum of LHCIIb and was clearly enriched in Chl b absorption
relative to PSII BBY particles (Fig.
3A). The fluorescence emission
spectrum of the A-band was broader, red-shifted and of lower yield
(approximately 50%) compared with LHCIIb (Fig. 3B). The
relative intensities of the vibronic satellite bands around 720 and 740 nm were also enhanced in the A-band.

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Fig. 3.
Absorption and fluorescence spectra of the
A-band. A, absorption spectra of the A-band
(dotted line) in comparison with purified LHCIIb trimers
(solid line) and BBY particles (dashed line);
B, fluorescence emission spectra, normalized to maximum
intensity at 77 K for the A-band (dotted line) and LHCIIb
(solid line); C, absorption spectra of monomers
prepared from the A-band (dotted line) and from
solubilization of BBY particles as in Fig. 1 (solid
line).
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Solubilization of the A-band and further separation on a sucrose
gradient showed that the fraction was heavily enriched in LHCIIb (Figs.
2 and 4). At low DM concentrations, it
was found that 64% Chl and 70% of the violaxanthin in the A-band was
found in the trimeric LHCIIb fraction. This trimer had a xanthophyll cycle content of 22%. Negligible free pigment was observed. The absorption spectrum of the monomeric LHCII band at 0.43 M
sucrose was enriched in Chl b absorption around 650 nm compared with
the spectrum of the monomeric band found when BBY particles are fully solubilized (Fig. 3C). The latter is a mixture of the CP26
and CP29 (see Fig. 4), but the monomeric LHCII fraction obtained from the A-band therefore probably contains, in addition, some LHCIIb.

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Fig. 4.
SDS-polyacrylamide gel electrophoresis of the
various samples derived from thylakoids: whole thylakoids
(th); PSII BBY particles (BBY); LHCII
monomers (m), LHCII trimers (tr), and
PSII cores (core) obtained by solubilization of BBY
particles as in Fig. 1; A-band from BBY particles
(A/BBY); A-band from thylakoids
(A/th); A-band trimer (A/tr) and
A-band monomer (A/m) prepared by solubilization of
A-band as in Fig. 2. WM, molecular weight markers (in
thousands).
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The polypeptide composition of the A-band is shown in Fig. 4; several
bands in the 25-31-kDa region are present. The Lhcb1-3 polypeptides
of LHCIIb are shown, together with the polypeptides of CP26 and CP29.
The trimer isolated from the A-band showed the presence of the Lhcb1-2
polypeptides, and these polypeptides were also detected in the monomer
fraction. Densitometric estimates suggested that the Lhcb1-2
polypeptides accounted for more than 85% of the protein content in the
A-band.
Binding Affinity of Carotenoid to LHCII--
These data showed
that a major fraction of violaxanthin, mostly bound to LHCIIb, was
easily lost upon detergent solubilization of either PSII membranes or
oligomeric LHCII. Violaxanthin was clearly less tightly bound than Chl
to this complex. Conversely, violaxanthin appeared to be more stably
bound to monomeric minor LHCII. It was therefore decided to investigate
in more depth the ease with which violaxanthin might be removed during
detergent solubilization of LHCII, in comparison with the other bound
pigments. Isolated LHCII fractions were incubated in different
concentrations of DM and subjected to sucrose gradient centrifugation.
The amounts of different pigments in the LHCII fractions and free
pigment zones were then analyzed.
The results of this experiment using LHCIIb are shown in Fig.
5. Treatment with a range of DM
concentrations resulted in progressive loss of violaxanthin from this
complex (Fig. 5A). At the highest concentration, less than
10% of the violaxanthin remained in the trimer band, with nearly 70%
being released into the free pigment zone. DM treatment removed
approximately 4.0% Chl, highly enriched in Chl a, but more of the
carotenoid (the carotenoid/Chl ratio was 0.72 in the free pigment
fraction). In comparison with the original IEF sample, the free pigment
was depleted in neoxanthin and lutein but contained 20% violaxanthin
(Fig. 5B). The carotenoids bound to the trimeric LHCII band
contained less than 1% violaxanthin. Ten percent of the Chl was found
in a monomeric band, and it was of note that this complex had a
violaxanthin content of approximately 20% and a lower content of
neoxanthin compared with the trimer. It was estimated that the amounts
of lutein and neoxanthin removed from LHCIIb as free pigment by
treatment with DM were only approximately 10 and 5%, respectively.

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Fig. 5.
Carotenoid binding to LHCIIb.
A, distribution of violaxanthin in the free pigment and
trimeric and monomeric bands following treatment of IEF-prepared LHCIIb
with 0.2, 0.6, and 1.2 mM DM. Data are expressed as
percentage of total applied to the gradient; B, content of
lutein (L), neoxanthin (N), and violaxanthin
(V), expressed as percentage of total carotenoid for
IEF-prepared LHCIIb after treatment with 1.2 mM DM and
fractionated by sucrose gradient centrifugation. All data are the means
of at least five measurements.
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LHCIIb trimers can be more efficiently broken into monomers by
treatment with phospholipase (22). The carotenoid composition of
monomers was again different from trimers (data not shown). In
particular, again it was found that neoxanthin was 30% reduced, and
the violaxanthin content was over 20 times higher than for trimeric LHCIIb.
Broadly similar behavior was displayed by CP29 and CP26, although some
important differences were found (Fig.
6). The free pigment zones again were
enriched in carotenoid compared with the bands of purified LHCII (in
this case a monomeric band), and differed in carotenoid composition
(Fig. 6A). The free pigment zone of CP26 was depleted in
neoxanthin but enriched in both violaxanthin and lutein, compared with
the initial IEF complex. Approximately 20% of Chl was removed by
treatment with 0.2 mM DM, and this resulted in the loss of
about 30% violaxanthin (Fig. 6C). There was negligible loss
of either lutein or neoxanthin. Higher concentration of DM caused
extensive loss of Chl from CP26, so that it was not possible to attempt
to selectively remove any greater proportion of violaxanthin.

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Fig. 6.
Carotenoid binding to CP26 and CP29.
A and B, content of lutein (L),
neoxanthin (N), and violaxanthin (V), expressed
as percentage of total carotenoid for IEF-prepared CP26 (A)
and CP29 (B) after treatment with 0.2 mM DM and
fractionation by sucrose gradient centrifugation. IEF,
original IEF preparation; Fp, free pigment zone;
monomer, purified monomeric band on the gradient.
C and D, distribution of violaxanthin
(V) and Chl in the free pigment and monomeric bands
following treatment of CP26 (C) and CP29 (D) with
0.2 mM DM. Data are expressed as percentage of total
applied to the gradient. All data are the means of at least five
measurements.
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For CP29, the content of violaxanthin was almost the same in the IEF
preparation, the free pigment zone, and the purified monomer, although
there was some depletion in neoxanthin and enrichment in lutein in the
free pigment zone (Fig. 6B). A 20% loss in violaxanthin was
accompanied by an almost equivalent loss of Chl (Fig. 6D). As with CP26, higher concentrations of detergent removed Chl from the
complex quite readily.
Using the data obtained from treatment of the complexes with DM, an
index of relative binding strength was calculated for each of the
pigment types: the pigment content in the initial IEF fraction (as a
percentage of total pigment) divided by the pigment content in the free
pigment zone. This ratio was normalized on the value for lutein, the
one carotenoid for which a binding site within the complex has been
structurally determined. The rationale is that if all pigments are
bound equivalently, then the composition of the pigments released into
the free pigment fraction would be identical to the initial composition
of the complex. For the LHCIIb trimer, neoxanthin was held as tightly as Chl b (Table II) This affinity was 5 times higher than lutein, which was in turn 5 times more strongly bound
than violaxanthin, which was revealed to be the pigment most weakly
associated with the trimeric complex. For LHCIIb monomers, lutein,
neoxanthin, and violaxanthin are bound with approximately equal
efficiency, but less than Chl a and Chl b. For the minor complexes,
neoxanthin was again the most tightly bound carotenoid, but the
differences between the three carotenoids was much less. For CP26,
violaxanthin and lutein were bound with equal strength, as for LHCIIb
monomers. Violaxanthin was bound more efficiently than lutein to
CP29.
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Table II
Efficiency of pigment binding to light harvesting complexes of
photosystem II
The relative binding efficiency of pigments to each complex was
determined as the ratio of the amount of carotenoid in the complex
divided by the amount in the free pigment. DEP refers to whether
the complex was prepared from de-epoxidised (+) or epoxidised ( )
thylakoids. CP29 (thy) represents CP29 prepared from thylakoids
treated to induce maximum de-epoxidation. Lut, lutein; neo, neoxanthin;
vio, violaxanthin; zea, zeaxanthin.
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A similar analysis to that described above was carried out for LHCII in
which violaxanthin had been partially de-epoxidized to zeaxanthin. As
shown previously (8), the DEPS (de-epoxidation state of the xanthophyll
cycle carotenoid pool (zeaxanthin + 1/2antheraxanthin)/(violaxanthin + antheraxanthin +zeaxanthin))
varied among the different LHCII components (Table
III). For LHCIIb, the DEPS was around
40%, close to the overall value obtained for the PSII BBY particles in
these experiments. After treatment with DM, the trimer band exhibited a
DEPS of over 80%, but the monomer was less than 20%. These
differences are caused by the depletion of violaxanthin from the trimer
but the retention of zeaxanthin, and the converse for monomers. For CP26, the DEPS was almost the same as for LHCIIb, whereas for CP29 it
was the lowest, at only 15%. In order to increase DEPS to the maximum
level, thylakoids were incubated at low pH in the presence of ascorbate
(19). Here, an overall DEPS of 75-80% was observed. For CP29 isolated
from these thylakoids by IEF, the DEPS was 52%.
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Table III
Carotenoid composition of LHCII following induction of violaxanthin
de-epoxidation
Complexes were prepared from de-epoxidised thylakoids prepared from
light-treated leaves, except for CP29 (thy), for which the thylakoids
were treated in vitro to induce maximum de-epoxidation
state. DEPS is the de-epoxidation state (zca + 1/2ant)/(viol + ant + zea). For LHCIIb, data are from
the IEF-prepared trimer, the trimer obtained by detergent
treatment and subsequent sucrose gradient centrifugation as in Fig. 5,
and the trimer derived from the A-band as in Fig. 2. Carotenoid values
are the percentage of total carotenoid. Lut, lutein; neo, neoxanthin;
vio, violaxanthin; ant, antheraxanthin; zea, zeaxanthin; XC, vio + ant + zea.
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Analysis of the A-band prepared from thylakoids with a high DEPS
(70-80%) was carried out (Table III). The xanthophyll cycle carotenoid accounted for 25% of the total carotenoid, and the DEPS was
88%. The LHCIIb trimer fraction prepared from the A-band was found to
have a DEPS of 100% and the monomer fraction 79%. The difference
between the monomer and the trimer appeared to result from the loss of
violaxanthin from the latter, resulting in a lower xanthophyll cycle
content (15%), compared with 21% in the monomer.
The relative binding strengths of zeaxanthin to different LHCII
components is shown in Table II. For LHCIIb trimers, it was found that
zeaxanthin was significantly more tightly bound than violaxanthin. For
LHCIIb monomers, zeaxanthin and violaxanthin were both bound quite
strongly, with zeaxanthin having somewhat less affinity. Zeaxanthin was
also less tightly bound than violaxanthin to CP29. The violaxanthin
remaining bound to CP29 after de-epoxidation was more tightly bound
than the total violaxanthin pool, with an affinity equal to neoxanthin
and Chl, and greater than lutein. In contrast, zeaxanthin was rather
weakly bound to CP29.
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DISCUSSION |
The Oligomeric A-Band--
Most of the xanthophyll cycle pigment
associated with PSII was found in an oligomeric LHCII fraction (the
A-band). This fraction is larger than previously reported oligomeric
LHCII preparations (23) and is different from PSII "supercomplexes"
(24, 25), being deficient in PSII core proteins. The Chl a/b ratio
indicates that 80% of the Chl is bound to LHCIIb, in agreement with
the observations that 64% of the Chl was in trimeric LHCIIb, and
approximately half of the monomeric band was also LHCIIb, the remainder
being a mixture of CP29 and CP26. We estimate the A-band to be
approximately 600 kDa, based on the estimates for other bands on the
sucrose gradient (LHCII trimer, 100 kDa; PSII monomer, 236 kDa; PSII
dimer, 430 kDa; PSI monomer, 500 kDa). This is consistent with it
containing 5 LHCIIb trimers and 2-3 monomers of minor LHCII. The
fluorescence yield was less than for LHCIIb trimers; the broadening of
the fluorescence spectrum and increase in the relative contribution of
the vibronic satellite band suggests a change in Chl environment within
the oligomer that gives rise to fluorescence quenching (20, 26).
The Xanthophyll Cycle Content of LHCII--
The xanthophyll cycle
carotenoids were less tightly bound to LHCII than were Chl, lutein, and
neoxanthin. The binding affinities of violaxanthin and zeaxanthin also
differed. Differences in carotenoid binding strength are predicted
because they have different end-group orientation and polarity (18).
Gentle isolation procedures were needed to determine the amounts of
these pigments bound to LHCII, and the weakness of binding means that
it is impossible to exclude the possible distortion of results arising
from the migration of pigments between binding sites during the
solubilization procedure. The combination of the observed binding
stoichiometries in different fractions and the efficiencies of binding
strongly suggests that some previously published results have
underestimated the number of xanthophyll cycle carotenoids bound to
these complexes in vivo. In Fig.
7, the ratio of violaxanthin to
neoxanthin is presented for the range of LHCIIb preparations. Because
neoxanthin is so tightly bound to LHCIIb, with 1 molecule present per
complex, this ratio provides a good measure of the binding
stoichiometry. In purified LHCIIb, and samples obtained at higher DM
concentrations, this ratio is less than 0.5. However, more gentle
treatments lead to a higher ratio, with the maximum found for the
A-band of just over 1; most significantly, the trimer purified from the
A-band has a value between 0.9 and 1.0.

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Fig. 7.
Molar ratios of violaxanthin to neoxanthin in
a range of LHCIIb preparations. BBY, complete BBY
particle; IEF, IEF-prepared LHCIIb; 22, 16, 13, 8, and 5, trimers extracted from BBY particles with
different DM/Chl ratios as described in Fig. 1; TrA, trimer
prepared from the A-band as in Fig. 2; A, A-band prepared
from thylakoids as in Fig. 2.
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It is necessary to distinguish between tightly and loosely bound
xanthophyll cycle carotenoid. For LHCIIb, there is no tightly bound
violaxanthin, and almost all of the violaxanthin can be removed from
this complex, explaining earlier results that there was no violaxanthin
binding (7). The data in Fig. 7 suggest that there are 3 loosely bound
violaxanthin molecules per trimer. For CP26, the binding strength of
violaxanthin is rather similar to the value for monomeric LHCIIb;
therefore, we suggest that CP26 does not contain a tightly bound
violaxanthin but 1 molecule of loosely bound pigment. In contrast, CP29
has a violaxanthin content that is significantly higher than either
lutein or neoxanthin. There is clearly a population of violaxanthin
tightly bound to the complex, but in addition, there is an additional
loosely bound violaxanthin. The much higher binding strength of the
violaxanthin remaining after maximum de-epoxidation compared with the
value obtained for the fully epoxidized sample is evidence for the
existence of at least two binding sites, with only the violaxanthin at
the loose site available for de-epoxidation.
Therefore, CP26 and CP29 might together bind between 2 and 3 xanthophyll cycle carotenoids. If a similar stoichiometry is assumed
for CP24 (9), then the minor LHCII will account for a maximum of 3-4
xanthophyll cycle carotenoids. The upper limit to that associated with
LHCIIb would appear to be 3.0 per trimer. Assuming approximately 4-5
trimers per PSII unit, this would give another 12-15 xanthophyll cycle
carotenoids. These data then predict that the total number associated
with the PSII antenna would be 15-19 molecules per reaction center.
The measured carotenoid composition of BBY particles suggested a value
of approximately 15 molecules (Table I; see also Ref. 27), all of which
are recovered in the A-band.
One factor that must be taken into account when considering the
stoichiometry of xanthophyll binding to LHCII is that the carotenoid
pool sizes are variable across different plant species and within the
same species depending on growth conditions (4, 5). In spinach, the
xanthophyll cycle pool size is approximately 22% of total carotenoid,
whereas this may be as high as 30-35% in high light-grown plants and
as low as 15% in shade plants. The data presented in this paper
indicate that there may be an upper limit of 20 violaxanthin molecules
associated with PSII. This suggests that under light-limited
conditions, not all of the available xanthophyll cycle binding
sites on LHCII are occupied. Furthermore, observations made on
reconstitution of LHCII in vitro (28-31), as well as on
mutants lacking particular carotenoids (32, 33), indicate that the
carotenoid binding sites are not entirely specific and that variations
in carotenoid composition may result from changes in the occupancy of
these sites.
Multiple Carotenoid Binding Sites in LHCII--
To explain the
data presented here, we suggest that each LHCII monomer has four
distinct carotenoid binding sites (Table
IV). The structural model for LHCIIb
identifies two carotenoids bound within the complex, and these are most
likely two luteins (12). These sites are identified as L1 and L2. The
location of the neoxanthin binding site (N) is not known, but the fact
that it is a particularly tight binding site in LHCIIb trimers, but not
in LHCIIb monomers, suggests it faces the interior of the trimeric
structure. Although some caution should be applied in the application
of the LHCIIb structural model to the minor complexes CP29 and CP26
(9), it is more than likely that the bound lutein occupies the same site in all three complexes. For both minor complexes, there is probably only one lutein site occupied. In CP29, the other site appears
to be occupied by violaxanthin. This is essentially in agreement with
the observations made on reconstituted CP29, although in this work, it
was suggested that this site could be occupied by either violaxanthin
or neoxanthin (9). Given that there is clearly an additional
neoxanthin-specific site on LHCIIb, this seems unlikely. Therefore, we
propose that CP29 also has an N site, which can be occupied by either
neoxanthin or violaxanthin in order to explain the substoichiometric
binding of neoxanthin. For CP26, it is less clear which, if any,
carotenoid replaces the second lutein. We suggest that violaxanthin
does not occupy this internal tight site, which may therefore remain
vacant, and that CP26 has an N site that is occupied only by
neoxanthin.
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Table IV
Suggested occupancy of four putative carotenoid binding sites on
LHCII
Lut, lutein; neo, neoxanthin; vio, violaxanthin.
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The loose binding site for violaxanthin (V) is most likely on the
periphery of the complex, probably in equilibrium with the lipid phase.
Violaxanthin is removed at low detergent concentration and appears to
be lost during the previously published methods of LHCIIb purification.
The empirically determined binding affinity of this site is 1 order of
magnitude lower than Chl, lutein, or neoxanthin. When LHCIIb is
monomerized, the affinity of this site increases significantly. Thus,
the violaxanthin bound to CP26 is most likely bound to the V site, with
an affinity equivalent to this site on LHCIIb monomers. For CP29, the
binding stoichiometry of greater than 1 for violaxanthin suggests that
there is a second site on this complex; because this binding is
estimated to be 0.4 molecules or less, we suggest that this is bound to
the N site.
Availability of Violaxanthin for De-epoxidation--
Violaxanthin
de-epoxidase is bound to the lumen surface of the thylakoid membrane
(34). Recent studies suggest that violaxanthin has to be removed from
the complex, inserted into the active site of the enzyme, de-epoxidized
to antheraxanthin, released, and then de-epoxidized at the second
end-group to form zeaxanthin (35). It is unlikely that this occurs with
a carotenoid tightly bound to the L1/L2 site on the interior of the
complex. Again, this indicates that the violaxanthin bound to the L2
site in CP29 is not available for de-epoxidation. In contrast to CP29,
CP26 exhibits a high DEPS. It is therefore unlikely that violaxanthin in this complex is bound at the L1/L2 site. In studies of mutants lacking lutein, violaxanthin appears to replace the missing carotenoid, presumably becoming bound to the L1/L2 sites (32); this violaxanthin does not appear to be available for de-epoxidation. The conclusion that
the violaxanthin involved in the xanthophyll cycle, and therefore in
the control of nonphotochemical quenching, is peripheral is also
consistent with the observed effects of endogenous xanthophyll cycle
carotenoids on the structure and function of LHCIIb, CP29, and CP26
(17, 18, 21, 36).
The available violaxanthin appears to be associated with V sites on
LHCII, and the majority of these are on LHCIIb. Under conditions
leading to de-epoxidation, the higher affinity of the trimer V site for
zeaxanthin relative to violaxanthin, not found for the monomeric
complexes, predicts a higher relative concentration of de-epoxidized
pigment in LHCIIb. It has been suggested that only a small fraction of
the zeaxanthin pool may be needed for nonphotochemical quenching and
that this is associated only with the minor complexes (14). Although
this idea cannot be ruled out by the present data, the association of
the majority of the zeaxanthin pool with LHCIIb suggests that the site
of action of the xanthophyll cycle in controlling nonphotochemical
quenching is not confined to the minor complexes but is a
property of the whole antenna, including LHCIIb.