From the Department of Plant Biology, The Carnegie
Institution of Washington, Stanford, California 94305 and the
¶ Department of Microbiology and Immunology, Stanford Medical
School, Stanford University, Stanford, California 94305
Received for publication, September 22, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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There are five Synechocystis PCC6803
genes encoding polypeptides with similarity to the Lhc polypeptides of
plants. Four of the polypeptides, designated HliA-D (Dolganov,
N. A. M., Bhaya, D., and Grossman, A. R. (1995)
Proc. Natl. Acad. Sci. U. S. A. 92, 636-640)
(corresponding to ScpC, ScpD, ScpB, and ScpE in Funk, C., and
Vermaas, W. (1999) Biochemistry 38, 9397-9404) contain a
single transmembrane domain. The fifth polypeptide (HemH) represents a
fusion between a ferrochelatase and an Hli-like polypeptide. By using
an epitope tag to identify specifically the different Hli polypeptides,
the accumulation of each (excluding HemH) was examined under various
environmental conditions. The levels of all of the Hli polypeptides
were elevated in high light and during nitrogen limitation, whereas
HliA, HliB, and HliC also accumulated to high levels following exposure
to sulfur deprivation and low temperature. The temporal pattern of
accumulation was significantly different among the different Hli
polypeptides. HliC rapidly accumulated in high light, and its level
remained high for at least 24 h. HliA and HliB also accumulated
rapidly, but their levels began to decline 9-12 h following the
imposition of high light. HliD was transiently expressed in high light
and was not detected 24 h after the initiation of high light
exposure. These results demonstrate that there is specificity to the
accumulation of the Hli polypeptides under a diverse range of
environmental conditions. Furthermore, mutants for the individual and
combinations of the hli genes were evaluated for their
fitness to grow in high light. Although all of the mutants grew as fast
as wild-type cells in low light, strains inactivated for
hliA or hliC/hliD were unable to compete with wild-type cells during co-cultivation in high light. A mutant lacking
all four hli genes gradually lost its photosynthesis
capacity and died in high light. Hence, the Hli polypeptides are
critical for survival when Synechocystis PCC6803 is
absorbing excess excitation energy and may allow the cells to cope more
effectively with the production of reactive oxygen species.
Light serves as an environmental signal that regulates
physiological and developmental processes and provides energy that fuels the reduction of inorganic carbon. However, when photosynthetic organisms absorb excess excitation energy (more than can be used in
photosynthesis), the light energy can cause damage to the cell (3, 4).
There are several ways in which excess, absorbed, light energy can be
harmful to photosynthetic organisms. It can accumulate in
light-harvesting antenna complexes and reaction centers and promote the
formation of singlet oxygen, superoxides, and hydroxyl radicals, all of
which are highly reactive and potentially toxic. Reactive oxygen
species could modify proteins, lipids, and nucleic acids, ultimately
causing a loss of cell viability (5).
The photosynthetic reaction center polypeptide D1, or the 32-kDa
polypeptide, is particularly susceptible to damage as a consequence of
absorption of excess excitation energy (3, 6-8); this was first
recognized by Kyle et al. (9). The 32-kDa polypeptide, together with the D2 polypeptide, forms the heterodimeric reaction center of photosystem II that binds all of the redox components involved in photosynthetic charge separation. The rapid restoration of
photosystem II function following photodamage indicates the existence
of a tightly regulated repair system (3). Repair processes include the
degradation of damaged D1 polypeptide, de novo synthesis of
D1 on chloroplast ribosomes, processing of newly synthesized D1,
association of D1 with chlorophyll and its reaction center partner
(D2), and assembly of the heterodimeric complex with other photosystem
II polypeptides (3, 7, 10).
Both algae and vascular plants have evolved mechanisms for
photo-acclimation that favor survival in high light
(HL)1 (4, 11). These
mechanisms involve changes in the composition of light-harvesting
and/or reaction center pigment-protein complexes (reviewed in Ref. 12),
redistribution of excitation energy between the photosystems (state
transitions) (13, 14), and stabilization of photosynthetic membranes
(15). Plants have also developed the capacity to efficiently transform
excess absorbed light energy into heat, thereby dissipating the energy
in a harmless manner. This thermal dissipation is measured as quenching
of chlorophyll fluorescence or nonphotochemical quenching (NPQ) (4, 16, 17). NPQ is primarily a consequence of the operation of the xanthophyll
cycle, which is required for the generation of zeaxanthin in HL (4,
18-20). The PsbS polypeptide, which has four membrane-spanning helices
and shows homology to Lhc polypeptides, is also needed for NPQ (21). It
has been postulated that quenching of singlet excited chlorophyll
occurs by direct energy transfer to zeaxanthin (22). However, recent
evidence (23, 24) suggests that xanthophyll-dependent quenching is more likely the result of conformational changes within
the antennae complex (17, 26-28). Energy dissipation within the
reaction center itself (29, 30) and cyclic electron flow around
photosystem II that involves a low potential form of cytochrome b559 (31) may also contribute to
photoprotection. Finally, reaction centers that are rendered
nonfunctional via the absorption of excess excitation energy may
continue to dissipate absorbed light energy as heat and serve a
photoprotective role with respect to neighboring, functional
photosystem II reaction centers (32). Other acclimation responses
include the synthesis and recruitment of enzymes with antioxidant
function such as superoxide dismutase (33), catalase (34-36), and
ascorbate peroxidase (37, 38). Additionally, abundant soluble
antioxidants in the chloroplast such as ascorbate and glutathione can
act as quenchers of triplet chlorophyll and singlet oxygen (39).
One group of proteins that accumulates upon exposure of plants to HL is
the ELIPs, or early light-inducible
proteins. These were originally characterized as
polypeptides that transiently accumulated in etiolated seedlings of pea
and barley following HL treatment (40-44). This transient accumulation
also occurred when plants were exposed to blue light, suggesting a role
for the blue light photoreceptor in the induction process (45); other
studies suggest that phytochrome may be involved in ELIP expression (46). In addition, ELIPs accumulate transiently under a
variety of stress conditions (47-50) that would cause photoinhibition. This raises the possibility that the ELIPs function to protect plants
from photooxidative damage and that expression of ELIP genes
may be controlled by the redox state of the cell and/or the
accumulation of reactive oxygen species.
ELIP genes from a number of different organisms have been
cloned and sequenced (43, 51-53). Sequence comparisons have revealed that they are members of the chlorophyll a/b-binding protein
or Lhc superfamily of proteins (54). The ELIPs have three transmembrane helices (TMH I-III) that correspond to the TMHs of the Lhc
polypeptides (55). Although pigment binding by ELIPs has not been
directly demonstrated, all ELIPs contain conserved residues that could potentially bind chlorophyll a (55). Even though it has been suggested that the ELIPs function as "pigment-carrier" proteins involved in the turnover and/or redistribution of pigment molecules under conditions when photosystem II components are being rapidly degraded and repaired (47), the exact role of ELIPs under light stress
conditions is not clear. Recently, the Cbr protein of
Dunaliella was shown to be associated with light-harvesting
antenna complexes II and preferentially associated with specific
pigment-protein subcomplexes that contain high levels of lutein and
other xanthophylls (56).
Members of the Lhc gene family have also been identified that encode
proteins with one and two TMHs. In Arabidopsis, two
ELIP-like genes that encode thylakoid membrane polypeptides with two
TMHs (the proteins are called Seps, stress
enhanced proteins) were isolated (57).
Expression of Sep genes increased in HL but not during other
stress conditions. An ELIP-like protein with a single TMH has also been
isolated from Arabidopsis (58). These single TMH
polypeptides, designated Hli or Scp (1, 2), were first discovered in
cyanobacteria. The single TMH in these polypeptides resembles TMH I or
III of the Lhc polypeptides. Expression of the genes is strikingly
similar to that of ELIP genes, suggesting that they have
similar functions. There are five monocistronic hli genes on
the Synechocystis PCC6803 genome (59, 60) that compose an
hli multigene family (Ref. 2, CyanoBase); one of these represents a fusion with the ferrochelatase gene.
We have examined accumulation of the four Hli proteins (the
ferrochelatase was excluded) of Synechocystis PCC6803 under
several conditions that would result in the absorption of excess
excitation energy by the photosynthetic apparatus, and we have
investigated the phenotypes of hli deletion mutants. Our
results indicate that Hli polypeptides accumulate when cyanobacteria
are exposed to HL or other stress conditions and that they may form
distinct protein complexes in the thylakoid membranes. Furthermore,
mutants that cannot synthesize Hli polypeptides show growth
characteristics similar to that of wild-type cells in low light (LL)
but are unable to compete with wild-type cells during exposure to HL. A
strain deleted for all four of the hli genes gradually loses
photosynthetic function and dies following exposure to HL.
Culture Conditions--
Synechocystis PCC6803 was
cultivated in BG-11 medium (61) buffered with 10 mM TES, pH
8.2, at 30 °C. Cultures were bubbled with 3% CO2 in air
and illuminated with 40 µmol photon m
For HL treatments, cells in mid-logarithmic growth phase
(OD730 ~0.8) were diluted with fresh medium to an
OD730 of ~0.3. The cells (in 50-ml culture tubes) were
then placed in a temperature-controlled glass chamber (maintained at
30 °C) and exposed to 500 µmol photon m Mutant Construction--
To construct cell lines in which each
of the Hli polypeptides was tagged with the His6
epitope, coding regions of individual hli genes were cloned
in frame into the pQE expression vectors (Qiagen) (pQE-60 for ssl1633
(hliC); pQE-70 for ssr2595 (hliB), ssr1789
(hliD), ssl2542 (hliA)). Each hli
promoter plus coding region (with the C-terminal His6 tag)
was ligated sequentially to the 5 S t1t2
prokaryotic terminator, a drug-resistant cartridge, and the DNA
sequences downstream of each of the corresponding hli genes.
Fig. 1 shows a linear drawing of each
plasmid containing an epitope-tagged chimeric hli gene, and
the legend of the figure provides the sequences of the primers that
were used to make the constructs. Each of the chimeric genes was
sequenced to ensure that no errors were generated during gene
construction. The constructs were transformed into
Synechocystis PCC6803; the wild-type hli sequence
was replaced by the chimeric hli-His6
sequence.
Plasmids containing the hliA and hliB genes
interrupted by erythromycin and spectinomycin resistance cassettes,
respectively, were gifts from Wim Vermaas (Arizona State University).
The hliA gene was interrupted at a SalI site
located 72 base pairs downstream of the translation start site. The
hliB gene was interrupted at a SacII site located
12 base pairs downstream of its translation start site. These
constructs were generated by Funk and
Vermaas2 and generously given
to us. The gene disruptions were confirmed by
PCR.3 The plasmids in
which hliC and hliD were deleted
(
The plasmids containing the interruptions/deletions were transformed
into Synechocystis PCC6803, and transformants were selected on appropriate antibiotics. Single, double, and quadruple mutants (all
of the hli genes were either disrupted or deleted) were
constructed. Transformants were continuously subcultured until each
mutant line contained homoplasmic interruptions of hli
genes. Segregation of the altered gene(s) in each of the mutants was
monitored by PCR of isolated genomic DNA using specific primers
as follows: hliA, GATGGCTTGGGGAGCTTTAC at position
701,108-701,127 and GTGTTACAATAGTTAACATAG at position
701,375-701,395; hliB, CTCTTTTGGTCAACAGACTTGAC at position
982,862-982,884 and GCCCTGGTTCAGTAGATTGCTTG at position 983,198-983,220; hliC, ACTACAGGTACCCCAGGCCAG at position
1,141,750-1,141,770 and TGAAACCTGATGAATGACGACG at position
1,142,194-1,142,215; hliD, TTGGTGTGGCAATGGCTGGATG at
position 398,000-398,021 and ATTGTACGCAAGCAGCAATAAGC at position
398,369-398,391. Preparation of Synechocystis PCC6803 genomic DNA was as described previously (63).
Preparation of Thylakoid Membranes--
Cyanobacterial cell
pellets derived from cells grown to mid-logarithmic phase were
resuspended in thylakoid buffer (1/100 of the original culture volume)
which contained 20 mM MES/NaOH, pH 6.4, 5 mM
MgCl2, 5 mM CaCl2, 20% glycerol
(v/v), 1 mM freshly made phenylmethylsulfonyl fluoride, and
5 mM benzamidine HCl. The cell suspensions (0.4-0.6 ml)
were transferred to a chilled microcentrifuge tube with
approximately 0.5 ml of glass beads pre-wetted by thylakoid buffer and
broken in a MiniBeadBeater by six breakage cycles at full speed (30 s
for each cycle, followed by 3-5 min of chilling in ice water). After
centrifugation at 1,600 × g for 10 min to remove
unbroken cells and cellular debris, the supernatant was diluted
30-50-fold in thylakoid buffer, and thylakoid and cytoplasmic
membranes were pelleted at 4 °C by centrifugation (20 min, 40,000 rpm in a Ti 50.2 rotor). The membranes were washed once and resuspended
in thylakoid buffer (1 ml of buffer to 200 ml of the original culture
volume). Sucrose density gradient centrifugation (64) was used to
purify thylakoid membranes. Purified membranes were resuspended in 0.1 M sodium phosphate buffer, pH 7.5, containing 0.3 M sodium chloride.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analyses--
Solubilization of thylakoid membranes and SDS-PAGE were
performed as described by Peter and Thornber (65). Approximately 30 µg/lane membrane proteins were resolved by SDS-PAGE in a 10-16% polyacrylamide gel. Polypeptides were transferred onto nitrocellulose membranes (66), and immunodetection of polypeptides containing the
His6 epitope tag was performed using commercial antibodies, as recommended by the suppliers of the antibodies (Santa Cruz Biotechnology; Qiagen).
Concentrations of soluble polypeptides and thylakoid membrane
polypeptides solubilized by incubation in 2% SDS at 37 °C for 15 min were determined. Protein extracts were centrifuged at 16,000 × g for 2 min to remove insoluble debris; the supernatants
were diluted 10-fold with water, and the protein content was measured using BCA protein assay reagents (Pierce) according to the
manufacturer's instructions.
Gel Filtration--
A thylakoid membrane suspension at a
concentration of 0.6 mg of chlorophyll/ml was solubilized with a
surfactant mixture composed of 0.6% octyl glucoside and 0.6% decyl
maltoside, at 4 °C for 30 min. The material that remained insoluble
was removed by centrifugation at 26,000 rpm (Beckman TL 100 rotor,
~30,000 × g) for 20 min at 4 °C. The supernatant
(100 µl) was loaded onto a Superose column (type 6 HR 10/30, Amersham
Pharmacia Biotech) that was connected to an FPLC system (Millipore
Waters, model 650E). The column was pre-equilibrated with elution
buffer (0.1 M sodium phosphate, pH 7.5, 0.3 M
sodium chloride, 0.1% octyl glucoside, 0.1% decyl maltoside) and
eluted with the same buffer at a flow rate of 0.4 ml/min. Fractions
(0.4 ml) were collected, and the polypeptides in the fractions were
concentrated by precipitation by making the samples 10%
trichloroacetic acid.
Growth and Competition Experiments--
Growth of the cultures
was monitored as a change in optical density at 730 nm. Competition
experiments were performed at 30 °C under LL (40 µmol photon
m Fluorescence Measurements--
The yield of chlorophyll
fluorescence was continuously monitored using a
pulse-amplitude-modulation chlorophyll fluorometer (Walz) with a
pulse-amplitude-modulation 103 accessory, a water-jacketed cuvette, and a Schott KL 1500 lamp, which provided the actinic light.
The cells were diluted to a chlorophyll concentration of 2 µg
ml Methyl Viologen and Norflurazon Treatment--
To induce
oxidative stress artificially, LL-grown cultures that were in
logarithmic phase were diluted to an OD730 of ~0.05 with
fresh BG-11 medium. Methyl viologen (MV) or norflurazon were added to
the cultures to a final concentration of 0.5 µM for MV and 25 µM for norflurazon. The cultures were incubated in
low or intermediate (200 µmol photon m Accumulation of Hli Polypeptides during Growth in Different
Environmental Conditions--
The hliA transcript of
Synechococcus PCC7942 was shown to accumulate upon exposure
of cells to HL and nutrient limitation conditions (1). Both conditions
result in the excess absorption of light energy by the photosynthetic
apparatus. The hliA gene is similar to the four genes
encoding Hli polypeptides (or Scps) in Synechocystis PCC6803
(2). Table I summarizes the gene names,
the open reading frame designations as given in CyanoBase, and
the size of the different deduced polypeptides. Fig.
2A shows the combinatorial
alignments of the Hli polypeptides of Synechocystis PCC6803.
Fig. 2B is a dendrogram representing the grouping of the
four polypeptides into two distinct classes. HliA and HliB are most
similar, with identities of 87.1%, whereas HliC and HliD are not quite
as similar, with identities of 44.7%. We did not analyze the HemH
protein, which is a fusion between the gene encoding the ferrochelatase
and an hli-like gene.
To measure the relative levels of the Hli polypeptides in
Synechocystis PCC6803 cells following exposure to different
environmental conditions, we created four distinct
Synechocystis PCC6803 strains in which one of the four Hli
proteins was marked by insertion of the His6 epitope. The
sites of insertion of the epitope tags are shown in Fig. 1.
Monospecific antibodies directed against the His6 epitope
were used to examine the levels of the specific Hli polypeptides in
total membranes following exposure of the epitope-tagged strains to
different environmental conditions. We did not detect Hli polypeptides
in cells grown in LL on complete medium, unless solubilized membrane
proteins were enriched for Hli polypeptides by passage over a
Ni2+ affinity column (which binds to the His6
epitope; not shown). Therefore, although the Hli polypeptides are
present in cells maintained in complete medium under LL conditions,
they only accumulate to very low levels.
As shown in Fig. 3, all four of the Hli
polypeptides accumulated to high levels following exposure of cells to
HL. We estimate that there is a better than 10-fold increase in the
levels of HliA, HliB, and HliC polypeptides following 6 h of HL
exposure. The Hli polypeptides also accumulated under other stress
conditions. The levels of all of the Hli polypeptides increased upon
nitrogen starvation. Starvation for sulfur or exposure to chilling
temperatures led to the accumulation of HliA, HliB, and HliC; this
accumulation was comparable to that observed in HL. Interestingly,
following exposure of the cells to low temperature, two polypeptides
that exhibited immunoreactivity with antibodies against the
His6 epitope tag were observed in the
HliC-His6-tagged Synechocystis PCC6803 cell
line. One of these polypeptides had a similar mobility on SDS-PAGE to
that of the HliC that accumulated during other stress conditions, whereas the mobility of the other was slightly less. The
increased apparent molecular mass of the more slowly migrating species
may result from a specific modification of the protein. Preliminary
results3 using anti-phosphothreonine/tyrosine/serine
antibodies (Zymed Laboratories Inc. Laboratories)
suggests that the change in mobility is not a consequence of
phosphorylation of the polypeptide. The accumulation of the Hli
polypeptides was generally lower during exposure of cells to
Molecular masses of HliA and HliB, as estimated by SDS-PAGE, were
roughly equivalent to values predicted from gene sequences. In
contrast, the apparent molecular masses of HliC and HliD were considerably less and more, respectively, than the values predicted from CyanoBase information. The N-terminal sequence (see the legend of
Fig. 2 for the sequence) of purified HliC polypeptide from Synechocystis PCC6803 cells exposed to HL revealed that this
polypeptide initiates at a methionine that is 69 nucleotides downstream
of the translation start site that had been predicted from the
nucleotide sequence (CyanoBase). This smaller polypeptide was the only
product detected by SDS-PAGE, although we cannot rule out the
possibility that it resulted from a rapid and specific proteolysis that
is not blocked by the suite of protease inhibitors used during the isolation of thylakoid membranes. The slow migration of HliD during SDS-PAGE may reflect altered binding of the anionic detergent.
Kinetics of Hli Polypeptide Accumulation upon High Light
Exposure--
To define the kinetics of accumulation of the different
Hli polypeptides, we isolated total cellular membranes at various times
following transfer of different epitope-tagged strains to HL, and we
evaluated the levels of the specific Hli polypeptides using antibodies
against the His6 epitope tag. Western blot analyses of
total membrane proteins are shown in Fig.
4. The accumulation of the HliA and HliB
reached a maximum level within 1 h of transfer to HL. This level
was maintained for up to 6 h following the initial transfer, after
which the levels of these polypeptides gradually declined. HliC
exhibited a slightly slower rate of accumulation, reaching maximum
abundance at 3 h; this maximal level was maintained over the
entire 24-h period tested. The level of the HliD polypeptide was lower
than that of the other polypeptides. This polypeptide peaked in
abundance at 6-9 h following the onset of HL and rapidly declined
thereafter. These results suggest that all of the Hli polypeptides play
a role in the acclimation of Synechocystis PCC6803 to HL.
Temporal differences in polypeptide levels that are observed may
reflect the different requirements of cells as they develop long term
strategies for surviving HL conditions.
Reduction in Hli Levels following Transfer of Cells to Low
Light--
The ELIPs are rapidly degraded during recovery of cells
from excess excitation (69). Many of the characteristics of ELIPs are
similar to those of the Hli polypeptides. To investigate the stability
of the Hli polypeptides in LL, we transferred Synechocystis PCC6803 hli-His6-tagged strains that had been
exposed to HL for 6 h to LL and immunologically monitored Hli
polypeptide levels. Aliquots of cells at different times following a
return to LL growth conditions were used for thylakoid membrane
isolation. As shown in the Western blots of Fig.
5, the HliA and HliB polypeptides were
extremely unstable, and there is a loss of more than 80% of these
polypeptides within 1 h of transfer of cells to LL. The HliC and
HliD polypeptides are stable for the initial 3 h, after which they
are rapidly degraded. This delay in the reduction in HliC and HliD
levels coincides with a delay in the recovery of cell division and
accumulation of phycocyanin and chlorophyll, suggesting that HliC and
HliD may be important during this "latent" recovery stage.
Interestingly, only when the Hli polypeptides were barely detectable
did cell division and pigment accumulation proceed.
FPLC Fractionation of Hli Complexes--
The Hli polypeptides were
demonstrated to be exclusively in the thylakoid membranes (data not
shown). To determine if they were constituents of multisubunit membrane
complexes or were functional as monomers, we isolated thylakoids from
the His6-tagged cell lines grown in HL for 6 h,
solubilized the membranes with non-ionic detergents, fractionated
membrane-protein complexes by FPLC, and tracked His6-tagged
Hli polypeptides using the epitope-specific antibodies. As shown in
Fig. 6, HliA and HliB polypeptides
co-eluted in the ~100-kDa fraction, whereas HliC and HliD co-eluted
in the ~50-kDa fraction. These data suggest that the Hli polypeptides function as complexes in the thylakoid membranes and that pairs of the
Hli polypeptides may be associated with each other.
High Light Sensitivity of hli Mutants--
We constructed
Synechocystis PCC6803 strains in which the hli
genes were inactivated by insertion of a drug-resistant marker gene;
single, double, and quadruple mutants were constructed (see Fig. 1 and
under "Materials and Methods"). The hliA,
hliC, and hliD are monocitronic (2);3
therefore, interruption or deletion of these genes should not have a
polar effect on downstream sequences. The hliB gene is co-transcribed with the open reading frame slr1544 that encodes a
hypothetical protein of 103 amino acids.3 Interruption of
hliB may have a polar effect; however, interruption of the
hliB gene does not have any observable phenotype under the
conditions tested. The relationship of the co-transcribed sequence to
the hli genes and its potential role in acclimation needs
further analysis.
To determine the fitness of the hli deletion strains to
compete with wild-type cells in HL, we performed competition
experiments in which the wild-type and mutant strains were mixed,
placed in HL, and samples of the culture taken at various times
following HL exposure to determine the wild-type:mutant ratio in the
cultures. When single mutants, double mutants, and the quadruple mutant in the hli genes were mixed with wild-type cells, the ratio
of mutant to wild-type cells remained constant during growth in LL for
at least 4-6 days. Furthermore, the hliB, hliC,
and hliD single mutants appeared to have none or little
competitive disadvantage in HL relative to wild-type cells (data not
shown). In contrast, when either the hliA or the
hliA/hliB and the hliC/hliD
double mutants were mixed with wild-type cells and exposed to HL, the ratio of wild-type to mutant cells rapidly increased. Four days following the initiation of HL exposure, the cultures contained 10% or
less of the mutant cells (Fig. 7). The
quadruple mutant was very sensitive to HL; its photosynthetic capacity
was reduced to a very low level within 12 h of the onset of HL
(Fig. 8A). After 2 days in HL
this mutant stopped dividing and gradually died (Fig. 8B).
These results clearly establish that the Hli polypeptide family is
required for the acclimation of Synechocystis PCC6803 to HL,
and also suggests that there is some redundancy in function of the Hli
polypeptides. Although all of the single and double hli
mutants can grow in HL, some cannot grow as well as wild-type cells. In
contrast, when all of the hli genes are disrupted, HL becomes lethal, although the strain grows at a rate comparable to that
of wild-type cells in LL.
Oxidative Stress Induction--
To test the sensitivity of the
hli quadruple mutant to artificially generated reactive
oxygen species, the growth of wild-type cells and the quadruple mutant
was monitored at a light intensity of 200 µmol photon
m We have used epitope tagging to study the accumulation of the
Synechocystis PCC6803 Hli polypeptides. All four of the Hli polypeptides are induced when the cyanobacterial cells are exposed to
stress conditions. They accumulate following nutrient limitation, cold
treatment, and high intensity illumination. Under all of these stress
conditions the cells absorb excess excitation energy that causes
hyper-reduction of the acceptor side components of photosystem II, the
formation of triplet chlorophyll, the generation of singlet oxygen
within antenna and reaction centers, and the production of superoxide
radicals (38, 72).
During growth of Synechocystis PCC6803 under our standard LL
conditions (40 µmol photon m Epitope tagging technology was used to examine the expression patterns
of the Hli polypeptides. Although this procedure is widely used and is
a generally accepted approach to examine expression and sub-cellular
location of proteins, there is the possibility that the
His6 tag alters the stability or aspects of targeting of
Hli polypeptides, which could affect expression patterns. To examine
this possibility we generated antibodies against recombinant HliD and
used the antibodies to quantify accumulation of HliD in both wild-type
cells and the hliD-His6 strain of
Synechocystis PCC6803. The patterns of accumulation of
His6-HliD and unmodified HliD following high light exposure
were identical.3 The kinetics of degradation of tagged and
untagged HliD were also identical.3 Although we have not
analyzed the pattern of expression of the other unmodified Hli
polypeptides, the results with respect to HliD make it unlikely that
the tag significantly influences expression patterns.
Interesting, Synechocystis PCC6803 stops growing immediately
following exposure to HL, and only resumes growth after approximately 6 h in HL. The initial accumulation of Hli polypeptides occurs during the phase of acclimation in which the cells are unable to
divide. Once the cells reach a new physiological steady state that
accommodates the new light conditions, they begin to divide and the
levels of Hli polypeptides fall. This decline may reflect a
modification of the polypeptide and lipid composition of the photosynthetic machinery that enables the cells to balance more efficiently the utilization and dissipation of absorbed light energy,
which allows for continued growth. These modifications also alter the
need for Hli polypeptides. The results also suggest that HliA, HliB,
and HliC may be important for sustained growth in HL and that these
polypeptides may have some overlapping function. This possibility is
supported by the finding that whereas the quadruple hli
mutant dies upon exposure to HL, none of the single or double mutants
die (although a number of them do not grow as fast as wild-type cells
following exposure to HL).
Some results suggest that HliA/HliB and HliC/HliD may form complexes in
the photosynthetic membranes. However, these results are only based on
co-migration of polypeptides following solubilization of thylakoid
membranes. The possibility of a complex between HliA/HliB has some
support with the findings that these polypeptides increase and decrease
with exactly the same kinetics following exposure to HL. Although the
kinetics of accumulation of HliC and HliD differ following the transfer
of cells from LL to HL, they decrease with the same kinetics following
transfer from HL to LL. In addition, the wild-type HliD polypeptide was
found to be present in sucrose gradient fractions containing
His6-tagged HliC.3 Hence, the Hli polypeptides
are likely to exist in multimeric structures, as suggested by the
migration of the solubilized polypeptides during gel filtration (Fig.
6) and their sedimentation in sucrose gradients. However, the nature of
these complexes and the structural relationships among the different
Hli polypeptides remain to be established.
A number of polypeptides have been identified that increase the ability
of photosynthetic organisms to survive HL exposure. Some of these
polypeptides such as superoxide dismutase and ascorbate peroxidase may
be involved in rapidly eliminating potentially toxic, reactive oxygen
species that form following HL exposure. Others such as PsbS (21, 73)
and IsiA (74) may be involved in quenching singlet excited chlorophyll
molecules, which would prevent the accumulation of toxic oxygen
species. The specific role of the Hli polypeptides in photoprotection
is still not clear, although it has been proposed to function in the
dissipation of excess absorbed excitation energy (1, 58) or serve as a
chlorophyll carrier (2).
A recent report by Funk and Vermaas (2) suggests that the
hli genes of Synechocystis PCC6803 are not
significantly induced when the cells are transferred from moderate
light (50 µmol photon m There are several lines of evidence to support the proposal that the
Hli polypeptides are required for survival and acclimation of cells to
the absorption of excess light energy and that they are probably not
major chlorophyll carriers in the cell (although they may be adapted to
bind and store free chlorophyll specifically when cells are absorbing
excess excitation). First, accumulation of Hli polypeptides is
triggered whenever Synechocystis PCC6803 is absorbing excess
excitation energy. Second, some of the single (hliA) and
double (hliC/hliD; hliA/hliB) mutants cannot
compete with wild-type cells during exposure to excess excitation
energy; their growth rate is equal to that of wild-type cells in LL
(doubling time of approximately 8 h). A mutant defective for all
four hli genes dies upon exposure to HL. When wild-type
cells are exposed to HL, cell growth stops and only proceeds after
approximately 6 h of acclimation. The cells then begin to rapidly
divide. Although the quadruple mutant grows at a similar rate to
wild-type cells even at light intensities up to 200 µmol photon
m The biggest questions that still remain are as follows. 1) How
widespread are the Hli polypeptides in photosynthetic organisms? 2) How
are they organized in the photosynthetic apparatus? 3) What are their
specific functions? 4) How do they perform these functions? 5) What are
the different specificities among the different members of this
polypeptide family in cyanobacteria? 6) What features of the different
polypeptides confer this specificity? Genes encoding Hli proteins have
been identified in a number of cyanobacteria (1, 60, 75) and red algae
(76-78). Recently, an hli cDNA was also identified from
Arabidopsis (58). Characterization of the
Arabidopsis hli gene suggests that the vascular
plant Hli polypeptide is imported into chloroplasts and, similar to
observations made with cyanobacteria, the level of the hli
transcript increases following exposure of Arabidopsis to HL
(58). These authors suggest that Hli polypeptides function in the
dissipation of excess excitation energy.
The work presented here demonstrates the accumulation of the different
Hli polypeptides in the thylakoid membranes and their requirement for
survival during exposure to HL. Furthermore, HL completely destroys
photosystem II function in the hli quadruple mutant,
suggesting that these polypeptides are involved in
protecting/stabilizing the photosynthetic apparatus, and perhaps other
aspects of the metabolic machinery of the cell, from photodestruction.
Although the precise mechanism for protection is still not clear, it is likely to involve either suppressed generation or elevated rates of
quenching of reactive oxygen species by pigment-protein complexes containing Hli polypeptides. This possibility is suggested by preliminary experiments in which cyanobacterial cells were exposed to
norflurazon. This inhibitor of carotenoid synthesis facilitates the
accumulation of singlet oxygen (79) and leads to the induction of the
Hli polypeptides in wild-type Synechocystis
PCC6803.3 Interestingly, the hli quadruple
mutant is significantly more sensitive to the administration of
sublethal doses of norflurazone than wild-type cells. The finding
suggests that the mutant strain has a reduced capacity for
detoxification of singlet oxygen. A more detailed biochemical analysis
of the wild-type and mutant strains should clearly establish the role
of the Hli polypeptides in maintaining photosynthetic activity and
viability of the cells in HL.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2
s
1 from incandescent bulbs. BG-11 medium
lacking nitrogen (
N) or sulfur (
S) was prepared by replacing the
NaNO3 for
N medium and MgSO4,
ZnSO4, and CuSO4 for
S medium with equimolar
amounts of the corresponding chloride salts (NaCl, MgCl2,
ZnCl2, and CuCl2, respectively). For nutrient
starvation experiments, cells grown in BG-11 medium were pelleted by
centrifugation (5,000 × g, 5 min) and re-suspended in
N or
S medium. This step was repeated prior to allowing cells to
grow in
N or
S medium. Procedures for initiating nutrient
deprivation have been described previously (62).
2
s
1 white light for various lengths of time,
as indicated in the text. For cold treatment, cultures were diluted
with BG-11 medium chilled to 4 °C and then allowed to incubate at
4 °C with constant shaking for 6 h.
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Fig. 1.
A, plasmid constructs showing the
different hli genes containing sequences encoding the
His6 epitope tag; and B, constructs showing the
deleted hliC and hliD genes. The primers
used for PCR amplification of the different hli genes
are as follows: hliA (0.23 kb),
CTCGGCCTATTCTAagat(CAGG)CTATTTAACCAACCAATG (701,127-701,162) and
TAATCCAAgc(TT)ATGCCCACCCGTGGCTTC (701,333-701,360); hliB
(0.23 kb), CAACAgc(CT)ATGc(A)CTAGCCGCGGATTT (982,961-982,985) and
ACCCAGCag(CA)ATct(TA)GAGAGAGAGCAACCAAC (983,161-983,190);
hliC (0.23 kb), CTATGGAAAg(A)ATct(TA)CAGAATGCCGAAGAAGTG
(1,141,792-1,141,823) and ACAGACTTGCCATGGGCGCAAT
(1,142,005-1,142,026); hliD (0.20 kb),
AGGAAATCg(C)CATGc(A)GTGAAGAACTACAAC (398,178-398,206) and
AAGCCAACTCCag(CT)At(G)Ct(G)CAGTCCCAACCAG (398,343-398,372). The
primers used to amplify the upstream (upstr) and downstream
(dwstr) sequences are as follows: hliA-dwstr
(0.49 kb), TTAGTTTGAg(T)CTc(A)TATTCCTTGCC (700,657-700,680) and
AAATAGCCTc(G)TAGAATAGGCCGAG (701,127-701,150); hliB-dwstr
(0.40 kb), TCTCTCTCTAga(AT)TGGCTGGGTGCAT (983,170-983,194) and
ACCGTGGCTga(TG)GCTCTGCTTACGG (983,553-983,576); hliC-dwstr
(0.38 kb), TTCCCAGAGctc(GGA)ACTGGCCGACGC (1,141,430-1,141,453)
and GGCATTCTag(GT)AATTTTTCCATAGTTC (1,141,789-1,141,814);
hliD- dwstr (0.42 kb), GCGCTAtct(GGG)AGa(T)TGGCTTATTGCTGCT
(398,355-398,381) and TTGAAGGag(CA)CTCCCGCCCAGATGG (398,749-398,772);
hliC-upstr (0.30 kb), CGGGAGGATcc(AA)TGTTAGGCTCAAAC
(1,142,041-1,142,065) and CCGTAGATa(+)Tc(+)GG TTACCAGTCTTTC
(1,142,311-1,142,334); hliD-upstr (0.38 kb),
TCGGGTGATa(+)TCAGCAGGAGTTGG (397,804-397,826) and
CCTGGGATcc(AA)TTAACTTAGTT TAC (398,157-398,180). The size
of each PCR fragment generated by the primer pair is given in the
legend next to the gene name in parentheses. Lowercase
letters in the primer sequences of the legend indicate mutations
introduced into the sequence; the original nucleotides are included in
parentheses to the left of the corresponding mutations. + designates an insertion. Numbers in the
parentheses to the left of each primer presented
in the legend indicate the primer position in the cyanobacterial genome
as given in CyanoBase. In the figure, the black boxes
designate a His6 tag. 5Stlt2 represents the 5 S
terminator sequence. The cassettes containing genes for drug resistance
are as follows: Cmr, 0.85 kbp, chloramphenicol resistance;
Kmr, 1.2 kbp, kanamycin resistance; Spcr, 2.0 kbp spectinomycin/streptomycin resistance. Restriction enzymes are
designated Bm, BamHI; Ev,
EcoRI; Hc, HincII; Hd,
HindIII; Sc, SacI; Sp,
SphI; Xh, XhoI.
hliC and
hliD) were generated by ligating
a PCR fragment upstream of each gene (0.3 kb for hliC; 0.4 kb for hliD), a drug-resistant cartridge (kanamycin for
hliC; chloramphenicol for hliD), and a PCR
fragment generated to sequences downstream of each gene (0.4 kb for
hliC; 0.5 kb for hliD), all in the proper
orientation. Primers used for PCR amplifications, given in the legend
of Fig. 1, incorporated different restriction endonuclease sites to
facilitate cloning. A detailed representation of the constructs is
depicted in Fig. 1.
2 s
1) or HL (500 µmol photon m
2
s
1). Wild-type Synechocystis
PCC6803 cells and mutant strains were mixed at approximately equal
densities (OD730 ~0.6) and diluted to an
OD730 of approximately 0.05. An aliquot (5 µl) of the
culture was diluted in 0.5 ml, and 50 µl (about 400 cells) was spread onto each BG-11 agar plate, both with and without antibiotics, to
determine the initial proportion of wild-type and mutant cells. The
mixed cultures were diluted ~10-fold with fresh medium containing appropriate antibiotics when the OD730 of the
culture approached 0.8. Aliquots from the culture were sampled at
various times following the initiation of the experiment and diluted, and approximately 400 cells were spread on each plate either containing or lacking the appropriate antibiotic.
1 prior to analysis. The minimal
fluorescence level (F0) was monitored with
red-modulated light (1.6 kHz) at 0.030 µmol photon
m
2 s
1. The maximum
fluorescence level of dark adapted (Fm) or
light-adapted (Fm') cells was assessed by a 600 ms high intensity white pulse at 3400 µmol photon
m
2 s
1. This light
pulse transiently closes all of the photosystem II reaction centers
(67). The maximal fluorescence level of a sample was determined in the
presence of 20 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (68) and white light.
2
s
1) light, and OD730 was used to
determine the rate of growth of the cultures at various times following
the addition of the herbicides.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Genes encoding Hli polypeptides of Synechocystis PCC6803
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Fig. 2.
A, alignment of the amino acid sequences
of the Hli polypeptides in Synechocystis PCC6803. Identical
amino acids are highlighted with a black
background, and similar amino acids have a shaded
background. Based on the N-terminal sequence of purified
Synechocystis PCC6803 HliC, which was MNNENSXF
(where the X is ambiguous), the initiation of translation is
at the second rather than the first methionine predicted in CyanoBase.
B, dendrogram constructed from the sequences shown in
A. The Hli polypeptides can be placed into two highly
related groups.
N and
S conditions, as compared with HL. Of all of the Hli polypeptides,
HliC accumulated to the greatest extent during
N and
S conditions
(approximately 55 and 65% of total Hli polypeptides, respectively,
under the conditions shown). HliD accumulated to the least extent under
all conditions tested and could not be detected when cells were exposed
to either
S or low temperature conditions.
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Fig. 3.
Accumulation of HliC, HliA, HliD, and HliB
polypeptides during various stress conditions. Cell membranes were
isolated and analyzed for Hli polypeptides that were
His6-tagged both before ( ) and after (+) the cells were
exposed to HL (HL, 6 h), low temperature (LT, 4 °C,
6 h), and nitrogen (
N) and sulfur (
S)
(62) deprivation for 12 and 30 h, respectively. Total membranes
were isolated as described under "Materials and Methods" and
membrane polypeptides fractionated by SDS-PAGE (12-16%
polyacrylamide). The polypeptides were blotted onto nitrocellulose
paper and probed with commercial antibodies specific for the
His6 tag (Santa Cruz Biotechnology). Control samples (
)
were maintained at standard growth conditions.
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Fig. 4.
Accumulation of HliC, HliA, HliD, and HliB
polypeptides following HL treatment. Wild-type (W) and
the His6-tagged cell lines (H) were placed in HL
for 1, 3, 6, 9, 12, and 24 h. At each time point the cells were
pelleted by centrifugation and disrupted using the MiniBeadBeater (as
described under "Materials and Methods"). The membranes were
isolated and analyzed for the His6-tagged Hli polypeptides
as described in the legend of Fig. 3.
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Fig. 5.
Change in the level of Hli polypeptides
following transfer of the cells from HL to LL. Cells were
incubated in HL for 6 h prior to being transferred to LL. The
membranes were isolated and analyzed for the His6-tagged
Hli polypeptides as described in the legend of Fig. 3.
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Fig. 6.
Fractionation of Hli polypeptides. Cells
containing the His6-tagged Hli polypeptides were incubated
in HL for 6 h. Membrane isolation was as described in the legend
of Fig. 3. The membranes were solubilized with mild detergents and
fractionated by fast protein liquid chromatography as described under
"Materials and Methods. Each fraction was analyzed for the
His6-tagged polypeptide as described in the legend of Fig.
3.
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Fig. 7.
Bar graphs representing the
competitive growth of wild-type cells (wt) and strains
inactivated for specific hli genes. The
hli mutant cells (hliA,
hliC/hliD, and hliA/hliB)
were mixed with wild-type cells, and the cultures were placed in HL
(right graphs) or LL (left graphs). Aliquots of
the cells were removed at the number of days indicated below each
bar in the bar graphs and plated onto BG11 agar plates with or
without the appropriate antibiotics. Plated cells were grown in LL for
2 weeks. Single colonies that formed on the plates were quantified to
determine the proportion of wild-type and hli-disruption
strains in the cell population.
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Fig. 8.
Effects of HL on variable fluorescence
(A) and cell growth (B).
LL-grown cells were diluted to a chlorophyll concentration of 2 µg/ml, transferred to a water-jacketed cuvette, and continuously
illuminated with HL. The chlorophyll fluorescence was continuously
monitored using a pulse-amplitude-modulation fluorometer as
described under "Materials and Methods." Growth of wild type
(wt) and the quadruple hli mutant in HL was
monitored as a change in optical density at 730 nm. Viability of the
cells was determined by spotting culture aliquots onto BG11 plates,
which were then incubated in LL for 1-2 weeks.
2 s
1 in the
presence and absence of 0.5 µM MV or 25 µM
norflurazon; these doses of the herbicide were sublethal for
Synechocystis PCC6803. In photosynthetic organisms, MV
catalyzes the formation of O2
primarily at the acceptor side of photosystem I. In contrast, the
herbicide norflurazon promotes the accumulation of
1O2* within thylakoid membrane as it inhibits
biosynthesis of carotenoids, which are the dominant quenchers of
1O2* generated by the antenna pigments. As
shown in Fig. 9, an intensity of 200 µmol photon m
2 s
1
does not markedly inhibit the growth of the quadruple mutant, and MV at
a sublethal concentration of 0.5 µM inhibits growth of
both strains to the same extent. These results suggest that the
activities of detoxification enzymes such as superoxide dismutase, peroxidases, and catalases are not significantly affected in
hli mutants. In contrast, norflurazon was shown to inhibit
consistently the growth of the quadruple mutant to a greater extent
than that of wild-type cells, suggesting that the Hli polypeptides may
play a role, either directly or indirectly, in detoxification of
1O2* generated within thylakoid membranes. The
sublethal concentration of norflurazon used to retard growth of
Synechocystis PCC6803 was 25 µM, which is
substantially higher than the dose (0.5-5 µM) reported
to kill Synechococcus PCC7942 (70, 71).
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Fig. 9.
Growth of wild type (wt) and
the quadruple hli mutant (4xhli) in
the presence of 0.5 µM MV and
25 µM norflurazon
(NF). Cells were incubated in 200 µmol photon
m 2 s
1, and cell
growth was measured as a change in optical density at 730 nm. Curves
were generated by averaging the data obtained from three representative
experiments. Errors were within 5% for all points.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2
s
1, 30 °C, nutrient-replete), the Hli
polypeptides are not detected unless specific methods are used for
their enrichment. By using genetic lines expressing
His6-tagged Hli polypeptides, enrichment was achieved by
binding the His6 tag to a Ni2+-affinity column.
The results demonstrated the presence of all four Hli polypeptides even
under LL conditions.3 However, the accumulation of the Hli
polypeptides is markedly stimulated following exposure of the cells to
a variety of different stress conditions. Although HliA, HliB, and HliC
accumulated under all stress conditions tested, HliD was only detected
in cells exposed to HL or starved for nitrogen (it was not detected in cells starved for sulfur or exposed to low temperatures). Not only does
HliD generally show the lowest levels of accumulation, its expression
is also transient in HL; the level of HliD peaks between 6 and 9 h, and by 24 h the polypeptide can no longer be detected. These
results suggest that the acclimation process results in either the
destabilization of HliD and/or strongly reduces the rate of
hliD transcription relative to degradation. All of the other
Hli polypeptides can be detected for at least 24 h following exposure to HL, with the level of HliC remaining constant from 3 to
24 h and the levels of HliA and HliB decreasing significantly. The
results also suggest that the acclimation process can be divided into
distinct phases. During the initial phase there is a rapid increase in
Hli polypeptides, especially HliA and HliB. HliD appears to be the
least sensitive to the HL treatment; it is the only Hli polypeptide
that cannot be detected after 1 h of exposure to HL. The levels of
all of the Hli polypeptides peak between 6 and 9 h, after which
they begin to fall, except for HliC. The level of HliD polypeptide
falls most rapidly and is not readily detected after 24 h in HL,
whereas HliA and HliB fall to about 25% of peak levels.
2
s
1) to HL (250 µmol photon
m
2 s
1). However,
high level hli expression was observed in glucose-grown cells lacking photosystem I or lacking both photosystems I and II (2).
The authors suggest that the Hli polypeptides function to bind free
chlorophyll under such conditions and that they may not be responsive
to the redox conditions of the cell. The binding of chlorophyll and/or
chlorophyll intermediates could protect the cyanobacterium from the
potentially phototoxic effect of these free pigments. Some aspects of
these data are difficult to interpret. The HL intensities used may not
have been sufficiently high to induce the hli genes
(especially if the signal for their induction relates to the
accumulation of reactive oxygen species) and/or the treatment times may
have been suboptimal for detecting hli transcripts
(1).3 Furthermore, mutants devoid of the photosystems may
be aberrant in membrane structure/organization because of the absence
of major complexes within the membranes. It would be difficult to
predict the redox state of such cells or their tendency for generating reactive oxygen species; either direct or indirect methods would be
required to quantify the levels of such species.
2 s
1 (Fig. 9), it
only grows to a small extent following the transfer to 500 µmol
photon m
2 s
1. After
the mutant experiences the 6-h HL acclimation period, it exhibits slow
growth that ceases after about 30 h, at which time the cells are
nearly all dead. Furthermore, during the first 6-10 h of acclimation,
wild-type cells lose approximately 50% of their capacity for
photosystem 2 activity (the variable fluorescence declines by 50%);
the remaining activity is sustained during HL growth. In contrast,
photosystem 2 activity in the quadruple mutant declines to nearly zero
following 10 h in HL, suggesting the destruction of the
photosynthetic machinery in the mutant strain. If the Hli polypeptides
served as major chlorophyll carriers, the quadruple mutant would be
expected to be impaired in growth in LL and moderate light since they
would likely be required as the cells are synthesizing high levels of
chlorophyll and chlorophyll-protein complexes under such conditions.
Furthermore, bleached, nitrogen-starved cells also synthesize high
levels of the Hli polypeptides.3 When nitrogen is provided
to the starved cultures, the cells regain their pigmentation and the
Hli proteins disappear. However, the disappearance of these proteins
precedes re-greening of the cell; these kinetic features are not so
easy to reconcile with a major chlorophyll carrier function.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Wim Vermaas and Christiane Funk for making some of the hli mutants available and Dr. Lori Van Wassbergen for designing the primers used for cloning the hli coding regions. We are also grateful to Devaki Bhaya, Chung Soon Im, Chao Jung Tu, Ling Zhang, John Christie, Jeff Shrager, Dafna Elrad, Barb Sears, and Winslow Briggs for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Department of Agriculture Grant 97-35301-4575 (to A. R. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Plant Biology, The Carnegie Institution of Washington, 260 Panama St., Stanford, CA 94305. Tel.: 650-325-1521 (ext. 286); E-mail: qingfang@ andrew2.stanford.edu.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008686200
2 C. Funk and W. Vermaas, unpublished data.
3 Q. He, N. Dolganov, O. Björkman, and A. R. Grossman, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: HL, high light; NPQ, nonphotochemical quenching; ELIP, early light inducible proteins; TMH, transmembrane helices; LL, low light; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; PCR, polymerase chain reaction; FPLC, fast protein liquid chromatography; MV, methyl viologen; PAGE, polyacrylamide gel electrophoresis; kb, kilobase; kbp, kilobase pairs.
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REFERENCES |
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