Plant Cell Physiol. 38(1): 17-24 (1997)
JSPP © 1997
Irradiance-Dependent Changes in the Size and Composition of the
Chlorophyll a-b Light-Harvesting Complex in the Green Alga Dunaliella
salina
Ayumi Tanaka' and Anastasios Melis
Department of Plant Biology, University of California, Berkeley, Ca 94720-3102, U.S.A.
Irradiance-dependent adjustments in the size and composition of the Chi a-b light-harvesting protein complex
(LHC-II) were investigated in the green alga Dunaliella
salina. Cells grown under low irradiance (100 fanol photons m~2 s"1) had a low Chi a/Chl b ratio, and an abundance of cellular Chi and LHC-II apoproteins compared to
cells grown under high light intensity (2,200 fanol photons
m~2 s"1). In low-light grown cells, four apoproteins of the
LHC-II migrating to 32, 31, 30 and 28.5 kDa were termed
according to their apparent molecular mass as LHC-II-1,
LHC-II-2, LHC-n-3 and LHC-II-4, respectively. In thylakoid membranes from high-light grown cells, LHC-II-1
was practically missing and LHC-II-2 was greatly depleted.
On a cell basis, the steady-state amount of total LHC-II
apoprotein in high-light grown cells, measured either from
the intensity of protein staining by Coomassie brilliant blue
or from Western blotting, was 5-7% of that in low-light.
In vivo rates of apoprotein biosynthesis were measured
from the time course of [3SS]sulfate incorporation into
LHC-II. On a cell basis, the rate of biosynthesis in highlight grown cells was about 20% of that in low-light grown
cells. Thus, in high-light grown cells, the rate of LHC-II
apoprotein biosynthesis was ~ 3 times greater than the
steady-state amounts of LHC-II present in thylakoids. The
results suggest discrete irradiance-dependent transcriptional and posttranslational regulation steps that define the
amount and composition of the LHC-II in chloroplasts.
growth. Photosynthetic organisms acclimate to the level of
irradiance by adjusting the size of the antenna associated
with each photosystem (Anderson 1986, Melis 1991, Lindahl et al. 1995). In PSII, the Chi a-b light-harvesting complex (LHC-II) is primarily responsible for the process of
light absorption. When plants are grown under low light
(LL) intensity, PSII contains a large LHC-II antenna, it
displays a low Chi a/Chl b ratio and a high LHC-II to
PSII-core ratio. When plants are grown under high light
(HL) intensity, PSII contains a small LHC-II antenna, it
displays a high Chi a/Chl b ratio and a low LHC-II to
PSII-core ratio (Bjorkman et al. 1972, Leong and Anderson 1984, Larsson et al. 1987, Lindahl et al. 1995).
In most green algae and higher plants, the LHC-II apoproteins are encoded for by several distinct genes (Lhcb
and Lhca for PSII and PSI, respectively) that belong to
the same multigene family (Dunsmuir 1985, Jansson et al.
1992). In D. salina, lhcb genes are encoded by a number of
homologous genes (Long et al. 1989, LaRoche et al. 1991).
Thus, heterogeneity in the LHC-II was reported at the level
of apoprotein (McDonnel and Staehelin 1980, Darr et al.
1986) and Chl-protein complex content (Camm and Green
1980, Tanaka et al. 1987, Peter and Thornber 1991). The
regulation of biosynthesis, assembly, macromolecular architecture, and association of the LHC-II with the PSIIcore complex is not known. The regulation of the size of
the LHC-II by irradiance, however, offers itself as a tool
through which to study the structure and hierarchy of
assembly of this Chi a-b thylakoid membrane protein complex.
Key words: Assembly — Light-harvesting complex — Protein composition — Photosynthetic unit — Thylakoid membrane — Dunaliella salina.
D. salina is a good model organism in which to study
the acclimation of the photosynthetic apparatus to irradiance. The cells acquire a high Chi a/Chl b ratio (over 20)
under high irradiance and a low Chi a/Chl b ratio of about
4 when grown under low irradiance (Smith et al. 1990). The
low Chi a/Chl b ratio under LL intensity is accompanied
by a significant increase in the cellular content of Chi (Harrison et al. 1992, Vasilikiotis and Melis 1994). The number
and composition of the LHC-II apoproteins also changed
in response to light intensity (Harrison et al. 1992, Webb
and Melis 1995). Similar results were also obtained in D. tertiolecta in which four distinct LHC-II apoproteins were resolved by SDS-PAGE (Sukenik et al. 1988, LaRoche et al.
1991). Their relative amounts in the chloroplast thylakoids
were reported to depend on the cell growth intensity.
Chl-protein complexes are major components of the
photosynthetic apparatus that serve to absorb light and to
transfer the excitation energy to photochemical reaction
centers (Thornber 1979). The amount and composition of
the Chl-protein complexes is variable in thylakoids, and
chiefly depends on the incident irradiance during plant
Abbreviations: LHC-II, Chi a-b light-harvesting complex of
PSII; LL, low light; HL, high light.
1
Permanent address: Department of Botany, Faculty of Science,
Kyoto University, Kyoto, 606-01 Japan.
17
18
Irradiance-dependent composition of the LHC-II
In this work, we investigated the content of LHC-II
apoproteins in D. salina which was grown under low and
high irradiance. D. salina exhibited significant changes in
Chi content per cell, Chi a/Ch\ b ratio, and in the amount
of cellular LHC-II. Rates of in vivo biosynthesis of LHC-II
apoproteins were compared with the steady-state amounts
of LHC-II apoproteins in the chloroplast thylakoids in
HL-grown and LL-grown cells. The results suggest discrete
irradiance-dependent transcriptional and posttranslational
regulation steps in the biosynthesis, assembly and composition of the LHC-II.
Materials and Methods
Cell growth—Dunaliella salina cultures were grown in an
artificial hypersaline medium similar to that of Pick et al. (1986)
containing 1.5 M NaCl, 5 mM MgSO4, 0.3 mM CaCl2, 0.1 mM
KH2PO4> 20 mM EDTA, 2 mM FeCl2, 5 mM NH4CI, and 40 mM
Tris-HCl, pH 7.5 supplemented with a mixture of micronutrients.
Carbon was supplied as NaHCO 3 added to the growth medium at
an initial concentration of 25 mM. Cultures were grown in flat bottles (optical path length = 3 cm) at 30°C under illumination at 100
A<mol photons m~ 2 s~' (low light, LL), 300|/mol photons m~2
s~' (medium low light), 700^mol photons m~ 2 s~' (medium high
light) or 2,200 /imol photons m~2 s~' (high light, HL). Cells were
grown to the late natural log phase prior to harvesting for the
measurements. The number of cells per ml of suspension was
counted using the improved Neubauer ultraplane and an Olympus
BH-2 light microscope with an amplification of 200 x .
[35S]sulfatepulse labeling of thylakoid membranes—To measure the in vivo rate of biosynthesis for LHC-II apoproteins,
D. salina cultures were pulse labeled with [35S]sulfate. Late logphase cultures, grown under 100 or 2,200 ftmo\ photons m~2 s~',
were harvested by centrifugation at 1,000 x g for 2 min at 4°C and
resuspended in a growth medium without sulfate for 4 h in the
light to deplete stored sulfate. Cultures were labeled with 222 kBq
ml" 1 [35S]sulfate for 1, 2, or 3 h at 30°C under 100 or 2,200^mol
photons m~2 s" 1 . Cells from 150 ml aliquots of the respective culture were used for the LHC-II analysis.
Thylakoid membrane isolation—Thylakoid membranes were
isolated by sucrose density gradient centrifugation (Keegstra and
Yousif 1986). Cells were harvested by centrifugation at 1,500 x g
for 3 min. Pellets were suspended in a buffer containing 20 mM
Tris-HCl (pH 8.0), 1 mM aminocaproic acid, 1 mM aminobenzamidine, 0.2% polyvinylpyrrolidone and 15% (w/v) glycerol and
centrifuged at 1,500xg for 3 min. The pellet was resuspended in
the same buffer with the exception of 1.5% instead of 15% glycerol used, and centrifuged at 10,000 x g for 10 min. The pellet was resuspended in a buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM
EDTA, and 0.6 M sucrose using a tight fitting glass homogenizer.
The homogenate was diluted with 2 volumes of 20 mM Tris-HCl
(pH 7.5), 2 mM EDTA and loaded on a 0.5-1.5 M sucrose linear
density gradient with 2 M sucrose cushion in a buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM EDTA and centrifuged at
100,000xg for 2 h with an SW 41 rotor at 4°C. The green band
was collected, diluted with the same buffer and centrifuged at
100,000xg for 1 h. The pellet (purified thylakoids) was used for
the measurements below.
Thylakoid membrane protein analysis—Thylakoid membranes were solubilized in a buffer containing 125 mM Tris-HCl
(pH 6.8), 3.5% SDS and 10% glycerol. The proteins were separat-
ed electrophoretically in a gel containing 12.5% acrylamide without urea (Laemmli 1970). The gel lanes were loaded with 2 nmol
Chi for staining, 0.2 nmol Chi for Western blotting, or with the extract of 6x 106 cells for labeling experiments. After electrophoresis, gels were stained with Coomassie brilliant blue.
Chi determination—Cells or thylakoid membranes were homogenized in 80% acetone and centrifuged at 10,000 x g for 10
min. The absorbance of the supernatant at 663 and 645 nm were
measured by a Shimadzu UV-160A spectrophotometer. The concentration of Chi was determined according to Arnon (1949).
Immunochemical analysis—Identification of LHC-II apoproteins was accomplished with immunoblot analysis using specific
polyclonal antibodies against the spinach LHC-II apoproteins.
After electrophoretic transfer of the SDS-PAGE resolved thylakoid membranes, the nitrocellulose filter was incubated with a
small amount of the LHC-II immune serum. The cross reaction between the LHC-II apoproteins and the polyclonal antibodies was
visualized by alkaline phosphate-conjugated antibodies (Smith et
al. 1990).
Quantitation of the LHC-II apoproteins—Coomassie stained
gels or visualized nitrocellulose filters were scanned with an LKBXL laser densitometer and the apoproteins were quantified by
measuring the integrated areas of the corresponding bands. For
the [35S]sulfate labeling experiments, dry gels were exposed to Molecular Dynamics storage phosphor screens. The phosphor screens
were subsequently scanned and bands quantified with a Molecular
Dynamics Phosphorlmager.
Results
Chi content of cells grown under high and low irradiance—As reported previously in work from this laboratory, D. salina cell growth occurs with approximately the
same rate under LL (lOO^mol photons m~ 2 s~') and HL
(2,200^mol photons m~ 2 s~') growth conditions (Vasilikiotis and Melis 1994, Baroli and Melis 1996). However,
the Chi content and the Chi a/Chl b ratio of cells from the
two cultures was significantly different. At all stages of
growth, we measured constant 0.2 x 10~6 nmol Chl/cell
and a Chi o/Chl 6=25 : 1 in HL-grown cultures. Conversely, we measured constant 1.46 x 10" 6 nmol Chl/cell and a
Chi o/Chl 6 = 5 : 1 in LL-grown cultures. Thus, LL-grown
cells contained 7-8 times more Chi than HL-grown cells.
Chi a/Chl b ratios were also different, with this value being
higher by about a factor of 5 in the HL than in the LLgrown cells. Since Chi b is primarily a pigment component
of the LHC of PSII, it follows that a high Chi a/Chl b
ratio in HL-grown cells signifies a smaller light harvesting
antenna size for this photosystem.
Protein composition of thylakoid membranes in HLand LL-grown cells—To investigate the LHC-II protein
composition, thylakoid membranes were purified by sucrose density gradient centrifugation. The crude thylakoid
membranes obtained upon differential centrifugation of
the cell homogenate were loaded on a 0.5-1.5 M sucrose
density gradient. Figure 1 shows the separation profiles of
the sucrose density gradient. A single green band was obtained following the gradient centrifugation from both LL
Irradiance-dependent composition of the LHC-II
0.5M sucrose
1
HL
.5M sucrose
2M sucrose •
LL
Sucrose density gradient
Fig. 1 Sucrose density gradient centrifugation profiles of thylakoid membranes from HL and LL grown D. salina cells. Crude
thylakoid membranes were loaded on a 0.5-1.5 M sucrose linear
density gradient with 2 M sucrose cushion in a buffer containing
20 mM Tris-HCl (pH 7.5), 2 mM EDTA and centrifuged at
100,000 x g for 2 h in a SW 41 rotor at 4°C.
and HL-grown cells. However, the thylakoids from the LL
cells had a higher density than those from the HL cells.
This difference is attributed to the greater extent of grana
HL
LL
LHCH-2
luicn-3
i'LHCII-4
If
formation in LL than in HL chloroplasts (Vasilikiotis and
Melis 1995). Yellowish-brown bands were found to accumulate at the interface between the 0.5 M sucrose and the sample loading zone. These pigments were much less abundant
in the LL than in the HL samples.
Figure 2A shows the SDS-PAGE polypeptide profile
of thylakoid membranes from LL and HL-grown cells.
Figure 2B shows in greater detail the relative intensity of
staining of proteins in the 26-35 kDa region. These results
show 4 distinct polypeptides that belong to the LHC-II
of D. salina. To facilitate presentation of the work, these
polypeptides were labeled as LHC-II-1 through LHC-II-4
(Fig. 2B). In our SDS-PAGE, they migrated with an apparent molecular weight of 32, 31, 30 and 28.5 kDa, respectively. The densitometric profiles of these proteins in LL
and HL-grown samples are shown in Figure 2C.
The identity of these four protein bands was further investigated by Western blot analysis with LHC-II specific
polyclonal antibodies (Fig. 3). The four bands (LHC-II-1
through LHC-II-4) were positively identified by the polyclonal antibodies. However, the four bands were consistently present only in the LL-grown samples. In the HLgrown samples, the LHC-II-1 band was faint or missing
altogether and the LHC-II-2 band was substantially decreased. These results suggest that LHC-II from LL-grown
samples contains at least four distinct polypeptides and
94.7-
HL
66.2-
LL
• LHCII-1
• LHCII-2
• LHCII-3
• LHCII-4
45.0 —
HL
31.0 —
21.5 —
m
B
LL
, - " LHCII-1
LHCII-2
^
LHCII-3
^ LHCII-4
14.5 kDa
Fig. 2 SDS-PAGE profiles of thylakoid membrane proteins
from HL and LL grown D. salina. Thylakoid membranes were purified by sucrose density gradient centrifugation, solubilized in a
buffer containing 3.5% SDS, and subjected to SDS-PAGE in the
absence of urea. Each lane was loaded with thylakoids proteins
from about 4 x 106 cells. Following electrophoresis, gels were stained with Coomassie brilliant blue (A). The area corresponding to
LHC-II apoproteins were amplified (B), and scanned with an
LKB-XL laser densitometer (C).
Fig. 3 Western blot analysis of thylakoid membrane proteins
from HL and LL grown D. salina cells. Each lane was loaded with
1.1 nmol of Chi. Proteins were transferred to a nitrocellulose filter
and reacted with specific polyclonal antibodies raised against the
LHC-II apoproteins. The cross-reactions were visualized by
alkaline phosphate-conjugated antibodies (A), and scanned with
an LKB-XL laser densitometer (B).
20
Irradiance-dependent composition of the LHC-II
Table 1 Relative LHC-II apoprotein content in LL and HL grown D. salina
Coomassie stained gels
HL-cells
LL-cells
Western blots
HL-cells
LL-cells
LHC-II
per cell
LHC-II
per Chi
Cell type
LHC-II-1
LHC-II-2
LHC-II-3
LHC-II-4
Total LHC-II
—
26.5
19.9
19.9
66.2
LHC-II-1
LHC-II-2
LHC-II-3
LHC-II-4
Total LHC-II
20.5
34.4
17.3
27.8
100.0
LHC-II-1
LHC-II-2
LHC-II-3
LHC-II-4
Total LHC-II
LHC-II-1
LHC-II-2
LHC-II-3
LHC-II-4
Total LHC-II
—
3.01
2.26
2.26
7.50
20.7
34.7
17.1
27.5
100.0
1.44
17.7
17.2
11.5
47.8
Chi b per
LHC-II
33
100
0.16
2.02
1.96
1.31
5.50
19.5
39.6
21.4
19.5
100.0
19.6
39.7
21.3
19.4
100.0
44
100
The apparent molecular weights of LHC-II-1, LHC-II-2, LHC-II-3 and LHC-II-4 are 32, 31, 30 and 28.5 kDa, respectively.
that HL-grown samples accumulate significantly lower
amounts of these LHC-II apoproteins (LaRoche et al.
1990, Harrison et al. 1992, Webb and Melis 1995).
Figure 2A also shows that certain other proteins were
present in elevated amounts in the HL-samples and lacked
in the LL-samples, or vice versa. The identity of these proteins was not investigated in this work.
Quantitation of LHC-II in HL- and LL-grown cells—
The amount of individual LHC-II apoproteins in HL- and
LL-grown cells was measured upon scanning Coomassiestained gels and nitrocellulose (Western blot) filters with a
laser densitometer. Similar results were obtained with these
two experimental approaches (Table 1). Total LHC-II per
HL-cell was 5-7% of that in LL-grown cells. On a Chi
basis, LHC-II in HL-grown cells was 45-65% of that in
LL-grown cells. These quantitative differences were not
uniformly applied among the four LHC-II polypeptides.
As evidenced in the results of Fig. 3 and Table 1, LHC-II-1
and LHC-II-2 were strongly attenuated by cell growth irradiance, whereas LHC-II-3 appeared to be largely independent of cell growth intensity. Table 1 also shows the relative
Chi b content in the LHC-II of LL- and HL-grown cells.
The Chi 6/LHC-II ratio is 2-3 times greater in the LLthan in the HL-grown cells. A discussion of the variable
Chi 6/LHC-II ratio among LL- and HL-grown cells is
mol photons nr 2 s" 1
100 300 700 2200
94.7—
66.2 —
45.0—
31.0 —
21.5 —
14.5 —
kDa
Fig. 4 SDS-PAGE profiles of thylakoid membrane proteins
from D. salina grown under different light intensities. Cells were
grown at 100, 300, 700 or 2,200/imol photons m~2 s" 1 . Each lane
was loaded with thylakoid membranes equivalent to 4.4xlO 6
cells. Following electrophoresis, gels were stained with Coomassie
brilliant blue.
Irradiance-dependent composition of the LHC-II
offered below.
mol photons nr 2 s- 1
100
300
700
2200
' LHCII-1
LHCII-2
LHCII-3
• LHCII-4
.3=3
1200
1000
21
LHCII-2
500
1000
1500
2000
Light intensity (|i.mol photons m"2s*')
Fig. 5 Western blot analysis of thylakoid membrane proteins
from D. salina grown under different light intensities. Each lane
was loaded with 0.25 nmol of Chi. Proteins were transferred to a
nitrocellulose filter and cross-reacted with antibodies against the
LHC-II. The cross-reactions were visualized by alkaline phosphate-conjugated antibodies (top panel), and quantitated with an
LKB-XL laser densitometer (lower panel).
LHC-II polypeptide content as a function of growth irradiance—The LHC-II polypeptide content was measured
in cells grown under several different light intensities. The
SDS-PAGE profile of thylakoid membrane proteins and
the LHC-II Western blots on nitrocellulose are shown in
Figures 4 and 5, respectively. Thylakoids from cells grown
at 100 and 300//mol photons m~2 s"1 exhibited quantitatively similar LHC-II protein profiles. Cells grown at 700
//mol photons m" 2 s"1 lacked LHC-II-1, had a significantly reduced amount of LHC-II-2 and somewhat lower
relative amounts of LHC-II-3 and LHC-II-4. Consistent
with the results shown in Table 1, the content of the total
LHC-II in the cell gradually decreased as the growth light
intensity increased.
Rate of synthesis of LHC-II in HL- and LL-grown
cells—To assess the in vivo rate of biosynthesis of LHC-II
apoproteins, we measured the time course of 35S incorporation into LHC-II apoproteins. Cells were incubated in the
presence [35S]sulfate (Vasilikiotis and Melis 1994), aliquots
were removed at 1, 2 and 3h, and thylakoid membranes
were isolated and purified by sucrose density gradient cen-
LHCII-3
_
—
•
—
LHCII-1
HL
LL
1
1.5
LHCII-2 ^
2
2.5
3
LHCII-3
LHCII-1
.
.
LHCII-4
1
Fig. 6 Autoradiogram of gel showing the distribution of (35S]-label as a function of incubation time in thylakoid membrane proteins of HL- and LL-grown D. salina. HL or LL D. salina cells
were incubated for 1, 2, or 3 h in the presence of [3!S]sulfate. Each
lane was loaded with thylakoids proteins from about 4 x 106 cells.
The dry gels were exposed to a Molecular Dynamics PhosphorImager screens.
1.5
2
2.5
3
Fig. 7 Quantitation of the distribution of [35S]sulfate among thylakoid membrane proteins. The rate of incorporation of [35S]sulfate into the various LHC-II apoproteins was calculated on the
basis of equal cell number in the HL and LL samples. For other
conditions, see legend to Fig. 6.
Irradiance-dependent composition of the LHC-II
22
trifugation. Thylakoid membrane proteins from 4xlO 6
cells were subjected to SDS-PAGE. 35S label incorporation
was visualized upon gel exposure to suitable X-ray films
(Fig. 6). The intensity of the label in the bands corresponding to the various LHC-II apoproteins was quantitated
upon scanning the X-ray negatives with a laser densitometer. Figure 7 shows that, on a per cell basis, the initial
(0-1 h) rate of biosynthesis of total LHC-II in HL-grown
was about 20% of that in LL-grown cells. A comparison
with the results in Table 1 shows that the above rate of
LHC-II apoprotein biosynthesis is ~ 3 fold greater than the
relative steady-state amount of LHC-II under HL-growth
conditions. The relatively high rates of LHC-II biosynthesis but low steady-state amounts of functional LHC-II protein in the thylakoid membrane of HL-grown cells suggest
an irradiance-dependent post-translational regulation in
the assembly of the LHC-II.
Discussion
D. salina cells contain, in addition to thylakoid membranes, an extensive biological membrane system. The latter may contaminate thylakoid membranes isolated by the
two-step differential centrifugation method. Comparison
of the protein profile of thylakoid membranes isolated by
sucrose density gradient centrifugation with those by direct
precipitation showed that several non-photosynthetic proteins may contaminate the crude thylakoid membrane fraction (not shown). A much larger amount of contaminant
proteins was observed in thylakoid membranes isolated
from HL-grown cells. This is attributed to the smaller
amount of thylakoids present in the HL-grown cells and to
the apparent lack of distinct grana in the latter (Vasilikiotis
and Melis 1995). Such contaminating proteins may interfere with the analysis of the LHC-II apoprotein composition in Coomassie stained gels, and with the determination
of the rates of the LHC-II biosynthesis as measured by the
rate of [35S]sulfate label incorporation. Contaminating proteins were not observed whenever the two-step centrifugation method was augmented by sucrose density gradient
centrifugation.
In D. tertiolecta, Sukenik et al. (1988) detected four
different LHC-II apoproteins (31, 30, 28.5, and 24.3 kDa)
and reported that the levels of the 31 and 30 kDa LHC-II
apoproteins declined by 75%, while the cellular level of the
28.5 kDa LHC-II apoprotein declined by less than 30%
when cells were grown under HL conditions. In D. salina
(this work), we report 5-7% of total LHC-II in HL-grown
compared to LL-grown cells. Four LHC-II apoproteins
were found, termed by us LHC-II-1, LHC-II-2, LHC-II-3,
and LHC-II-4, that migrated in the 32, 31, 30 and 28.5 kDa
region. In spite of the slightly different electrophoretic
mobilities, we suggest a correspondence between the LHCII apoproteins in D. salina (this work) and D. tertiolecta
(Sukenik et al. 1988). Of these, LHC-II-1 was almost entirely missing in HL-grown D. salina cells. Similarly, the
amount of LHC-II-2 was substantially reduced in the HLgrown samples. It is suggested that LHC-II-1 and LHC-II2 may be the most peripheral components of the LHC-II,
compared with the LHC-II-3 and LHC-II-4 which may occupy a more internal position in the supramolecular structure of the LHC-II. PS-II units deficient in LHC-II-1 and
LHC-II-2 would be expected to have smaller Chi antenna
size compared to those of LL-grown cells. This was indeed
confirmed in direct measurements of the functional Chi
antenna size in LL- and HL-grown D. salina (Smith et al.
1990).
In vivo biosynthesis measurements showed that, on a
per cell basis, LHC-II apoproteins accumulated much
slower in the HL- than in the LL-grown cells (Fig. 7). This
observation is consistent with the notion that expression of
the various LHC-II genes is regulated at the transcriptional
level by the intensity of the growth irradiance (LaRoche
et al. 1991). However, in addition to the transcriptional
regulation of the LHC-II genes, there appears to exist a fine
qualitative regulation in the assembly of the various LHCII apoproteins. Thus, the results of Fig. 7 (HL) show that,
initially, all LHC-II apoproteins were synthesized with
comparable rates. However, analysis of the steady-state
amounts of LHC-II in thylakoids (Table 1) revealed that
LHC-II-1 and LHC-II-2 were greatly depleted from the thylakoid membrane of HL-grown cells. One explanation is
that apoproteins of the LHC-II in D. salina are synthesized
with about the same rate but the eventual assembly of the
LHC-II is determined post-translationally by the stability
of the various LHC-II apoproteins. Stability can be determined by the amount of Chi available to the chloroplast
under the different growth irradiance conditions. This
explanation is consistent with independent observations
showing that Chi availability determines the hierarchy of
assembly of photosynthetic complexes (Greene et al. 1988a,
b, Eichacker et al. 1990, Melis et al. 1996). The differential
stabilization of LHC-II apoproteins by Chi would be one
important mechanism for the coordination of Chi and apoprotein accumulation in the thylakoid membrane of photosynthesis (Apel and Kloppstech 1980, Bennett 1981).
In higher plants, LHC-II has been considered to bind
a fixed number of Chi a and Chi b molecules. As a consequence, lack of sufficient Chi b in the chloroplast is
thought to prevent the proper folding of the LHC-II proteins, thereby leading to LHC-II degradation (Bennett
1981, Bellamare et al. 1982). However, in D. tertiolecta, it
was reported that the Chi a/Chl b ratio of isolated LHC-II
was variable and that this ratio changed in response to
growth irradiance (Sukenik et al. 1987). Similar results
were obtained in this study with D. salina in which the Chi
6/LHC-II ratio was 2-3 times greater in LL- than in HLgrown cells (Table 1). This result suggests that internal pro-
Irradiance-dependent composition of the LHC-II
tein constituents in the supramolecular structure of the
LHC-II contain less Chi b than peripheral ones. Alternatively, the LHC-II in HL-grown cells may not be fully
pigmented with Chi b. In a previous study (Shimada et al.
1990), similar observations were made with cucumber cotyledons where the amount of LHC-II apoproteins increased
somewhat more rapidly than the increase in the levels of
Chi b, especially during the early stage of cucumber cotyledon greening. These observations points to an important
mechanism for the acclimation of the photosynthetic apparatus to the prevailing environmental light conditions. It
is suggested that, under HL conditions, LHC-II would not
function efficiently due to the absence of Chi b from its
holoprotein. When cells encounter a LL environment, however, Chi b could be incorporated into existing LHC-II
simply by filling the vacant Chi b slots.
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(Received August 7, 1996; Accepted October 18, 1996)