Photosynthesis Research 65: 83–92, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
83
Regular paper
Circularly polarized chlorophyll luminescence reflects the
macro-organization of grana in pea chloroplasts
Eugene E. Gussakovsky1,2,∗, Yosepha Shahak1 , Herbert van Amerongen3 & Virginijus Barzda3
1 Institute of Horticulture, The Volcani Center,
P.O. Box 6, Bet Dagan, 50250, Israel; 2 Department of Life Sciences,
Bar Ilan University, Ramat Gan, 52900, Israel; 3 Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan
1081, 1081 HV Amsterdam, The Netherlands; ∗ Author for correspondence (e-mail:gussak@agri.gov.il; fax: +9723-9669583)
Received 12 February 2000; accepted in revised form 26 July 2000
Key words: circular dichroism, emission anisotropy factor, ionic strength, osmotic pressure, photoinhibition
Abstract
Circular polarization of luminescence (CPL; Steinberg IZ (1978) Annu Rev Biophys Bioeng 7: 113–137) was
applied to study pea chloroplasts in different structural states. The structural changes of chloroplasts were induced
by variation of osmotic pressure, concentration of magnesium-ions or photoinhibition. Both large CPL and psi-type
circular dichroism (psi, polymerization and salt induced) signals appeared in the presence of granal macrostructure and were sensitive to structural changes of the grana. The relation was studied between the amount of CPL
expressed as an emission anisotropy factor gem and amplitudes of the red psi-type CD bands. The positive psi-type
CD band was not directly correlated with gem possibly due to a large contribution of circular intensity differential
scattering to the measured CD spectra. However, a linear correlation between the amplitude of the negative psitype CD band and gem was found. The CPL signal of pea chloroplasts was attributed to a psi-type origin, which
is observed in macroaggregates with densely packed chromophores with a long-range chiral order, and directly
depends on the level of macroorganization. With the use of CPL-based microscopy, the long-range packing of
LHC II particles can be studied in individual chloroplasts in future. In addition, the CPL method in general allows
the study of the macro-organization of grana in green leaves, where conventional light-transmission methods fail.
Abbreviations: CD – circular dichroism; CIDS – circular intensity differential scattering; Chl – chlorophyll; CPL –
circular polarization of luminescence; DTT – dithiothreitol; Fv – variable fluorescence; Fm – maximal fluorescence;
LHC II – light-harvesting chlorophyll a/b pigment-protein complex; psi – polymerization and salt induced
Introduction
Photosynthesis is a process with a high quantum efficiency under low-light conditions, which is, to a large
extent, due to the high-absorption cross-section, rapid
excitation energy transfer to and fast charge separation (van Grondelle et al., 1994). Under light-stress
conditions, light-harvesting chlorophyll a/b pigment–
protein complexes (LHC II) adapt to variations in environmental conditions by different regulation mechanisms, usually via non-photochemical quenching
processes (Horton et al. 1994). These mechanisms can
involve light-induced LHC II structure alteration. On
the other hand, LHC II is able to form chiral macroaggregates of LHC II particles which were suggested
to be involved in long-range migration/delocalization
of the excitation energy in the granum membranes
and in regulating energy dissipation (Garab 1996; Istokovics et al. 1997). So, the regulation mechanisms
may be often accompanied by long-range structural
reorganization of LHC II.
Granal thylakoid membranes of chloroplasts exhibit strong anomalous non-conservative CD signals in
the chlorophyll Qy and Soret absorption bands (Garab
84
1996). These signals are about one order of magnitude
stronger than the CD signals of individual pigment–
protein complexes constituting the grana. The anomalous CD signals are sensitive to orientation, macroorganization and size of the grana (Garab et al. 1988a,
1991; Barzda et al. 1994). They exhibit reversible
changes under short (1–2 min) illumination conditions
(Garab et al. 1988b; Barzda et al. 1996; Istokovics et
al. 1997) and irreversible changes under longer photoinhibitory illumination (Gussakovsky et al. 1997).
The anomalous CD signals were also observed when a
confocal scanning differential polarization microscope
was used to image chloroplasts. The CD images revealed positive and negative signals emerging from
different regions of the chloroplast (Finzi et al. 1989,
1991). These observations led to attribute the anomalous CD bands of chloroplasts to a psi-type origin
(Garab et al. 1988a, 1991; Barzda et al. 1994).
Psi-type (psi, polymerization and salt induced)
CD theory (Keller and Bustamante 1988) stated that
psi-type CD could appear in large, densely packed
molecular aggregates in which the long-range organization of chromophores is significant. In such systems,
absorption of light was considered as a collective property of a large number of chromophores coupled to
each other via long-range interactions. According to
this concept, emission from such a system should
also be considered as a collective property of a large
number of chromophores. Thus circular polarized luminescence (CPL) when excited by a non-polarized
light (see below) is expected to give large signals for
psi-type aggregates.
The psi-type CD bands are always accompanied by
circular intensity differential scattering (CIDS). The
measured CD signal, appearing as the differential extinction of left (L) and right-handed (R) circularly
polarized light, consists of differential absorption and
differential scattering (Keller and Bustamante 1986).
In practice, it is difficult to distinguish the absorption determined CD and CIDS contributions to the
apparent CD signal. One possibility to estimate the
differential scattering contribution to the apparent CD
signal is to compare the signals of CD and CPL.
A chiral molecule that exhibits CD, is expected
to spontaneously emit circularly polarized light even
with non-polarized excitation (Steinberg 1978a,b).
CPL is expressed by the emission anisotropy factor,
gem which is the weighted difference between the leftand right-handed circularly polarized components of
the emitted light:
gem =
I m(A0 |p|B0 )(B0 |m|A0 )
=
|(A0 |p|B0|)2
1f
Ple − Pre
=
(Ple + Pre )/2
f /2
(1)
where p and m are the electric and magnetic dipole moment operators respectively, A0 and B0 are the
quantum states involved in the emission processes,
Ple and Pre are probabilities of left- and right-handed
circularly polarized emission, respectively, f and 1f
are the total luminescence intensity and its circularly
polarized part, respectively. When the left-handed portion of the circularly polarized emission is larger, the
gem -factor is obviously positive according to Equation (1). CD and CPL are complementary techniques.
CPL is sensitive to environmental changes in the excited state. More detailed information about the CPL
method is available in reviews (Steinberg 1978a,b;
Riehl and Richardson 1986).
Isolated chlorophyll (Chl) molecules (monomers)
only give rise to a small negative CD signal in the
Qy band (Houssier and Sauer 1970) and negligible
CPL signal (Gafni et al. 1975). However, the first
work on CPL of chloroplasts showed a big positive
CPL signal dependent on the Hill reaction activity of
chloroplasts (Gafni et al. 1975). Later it was reported
that photoinhibitory illumination, EDTA and dithiothreitol affected the CPL signal of lemon chloroplasts
(Gussakovsky and Shahak 1996).
In the present work, we studied the relation
between the gem -factor of the Chl fluorescence and
the CD signals for different macro-organizations of
the grana in pea chloroplasts induced by variation of
osmotic pressure, magnesium-ions or by photoinhibition. It was shown that the CPL signal appeared only
in the presence of macro-organization of the grana.
A close correlation between the negative psi-type CD
band in the Qy absorption region and gem was found.
The positive psi-type CD band was not linearly correlated with gem . The strong sensitivity of the CPL signal
to the changes in the grana organization opens a new
possibility for structural studies of macroaggregates in
samples of high density such as chloroplasts in intact
leaves where CD and other transmission methods are
met with large difficulties.
85
Materials and methods
Chloroplasts were isolated from pea leaves of 2-weekold plants by a standard method as described elsewhere (Garab et al. 1988a). The stock suspension of
chloroplasts of 2–3 mg Chl/ml in the standard buffer (0.4 M sorbitol, 20 mM tricine–NaOH of pH 7.6,
5 mM MgCl2 , 10 mM KCl) was used within 4–5 hours
after isolation. Chl(a+b) concentration was determined according to Arnon (1949). The Chl a/b ratio
was about 2.3–2.5. Absorption spectra were measured
with a model 17 DS Spectrophotometer (Aviv Assoc.,
Lakewood, New Jersey).
The same sample of chloroplast suspension was
used for the set of CD, CPL and modulated chlorophyll fluorescence measurements. To avoid a possible residual influence of the saturation pulses on
chloroplasts during modulated fluorescence induction
measurements, CD was measured first, CPL next and
modulated chlorophyll fluorescence last.
A maximal photochemical quantum yield of
chloroplast photosynthesis was probed via the Fv /Fm
(Fv and Fm stands for variable and maximal fluorescence, respectively) value of the modulated Chl
fluorescence measured with a PAM-2000 Chlorophyll
Fluorimeter (Walz, Germany) (Schreiber et al. 1994).
The irreversible photoinhibited state of chloroplasts was obtained by a stepwise illumination for 11
min with white light of about 3–4 mmol m−2 s−1 (as
measured with a Li-Cor quantum sensor LI-190SA)
passed through a heat filter of 10 cm water and infrared
glass. After illumination, the chloroplasts were darkadapted for 15 min. Chloroplasts were studied in the
standard buffer supplemented by 5 mM DTT. For control, a chloroplast suspension of 20 µg/ml was kept in
the dark for 15 min instead of the light and the rest of
the procedure was the same (total time about 40 min).
No significant changes in Fv /Fm were found for the
control sample.
CD was measured with a model 62A DS Circular Dichroism Spectrometer (Aviv Assoc., Lakewood,
New Jersey) calibrated by camphorsulfonic acid as described elsewhere (Woody 1975). The absolute error
of the ellipticity measurements was about ± 0.3 mdeg.
A 5 mM pathlength cuvette was used for both the CPL
and CD measurements. The Chl concentration was
adjusted to 20 µg/ml in all experiments.
CPL was measured with a home-built instrument
as described earlier (Gussakovsky and Haas 1995).
Briefly, it consists of an mercury lamp, double monochromator, lens and depolarizer, resulting in an incid-
ent beam for the fluorescence excitation, and cutoff
filter, elasto-optical modulator, lens, monochromator
and photomultiplier for the collection of the emitted light. The emission collection was at 180◦ to the
excitation beam. The preamplified photovoltage was
applied to a lock-in amplifier. The total photovoltage
(total fluorescence) and the lock-in amplifier output
voltage (circularly polarized luminescence) were fed
into an A/D converter for computer processing and
calculation of the emission anisotropy factor gem according to Equation (1). The Chls were excited at 436
nm. A red cutoff glass filter transmitting above 630
nm, was used in the detection branch of the setup.
The spectral resolution of the CPL measurements was
about 20 nm.
The CPL instrument was calibrated using (1R)(-)-camphorquinone (Aldrich) in chloroform (Luk and
Richardson 1974; Schauetre et al. 1995) which was
found to have a gem -value of –7.1×10−3 in the 490–
530 nm range. This value was considered to be equal
to the absorption anisotropy factor gab of the same
solution calculated as
gab = 1A/A
where 1A and A are the CD and optical density values
at 436 nm (the excitation wavelength), respectively.
This was based (Steinberg 1978a) on the fact that both
gem and gab do not vary over the fluorescence and
red absorption bands, respectively. The error of the
gem measurement was about 5% at values higher than
about gem = 10×10−4 but the absolute error was not
smaller than 0.5×10−4.
The CPL signal of the chloroplast suspension was
measured at room temperature (about 20–25 ◦ C) and
found to be independent of the intensity of the excitation light in the range of 10–250 µmol m−2 s−1
measured with a Li-Cor quantum sensor LI-190SA.
Results
The CPL and CD spectra of pea chloroplasts in
different media
Figure 1 shows the CPL spectra of pea chloroplasts
in media of different compositions causing variations
in the stacking of the grana. All the spectra had a
maximum at about 690 nm. The intensity vanished
at 650 nm, but remained constant in the 730–760
nm wavelength range. The shape of the CPL spectra
was similar to those reported for lettuce chloroplasts
(Gafni et al. 1975).
86
Figure 1. The CPL spectra of pea chloroplasts in the standard
medium (1, open circles), in 20 mM tricine–NaOH pH 7.6 supplemented with 0.4 M sorbitol and 5 mM MgCl2 (2, triangles), in
20 mM tricine–NaOH pH 7.6 supplemented with 0.4 M sorbitol (3,
open rhombs), in 20 mM tricine–NaOH pH 7.6 supplemented with
5 mM MgCl2 (4, filled rhombs). The spectrum (5, filled circles)
represents chloroplasts in 20 mM tricine–NaOH, pH 7.6 without any
supplements. See ‘Materials and methods’ for details.
The amplitudes of the CPL signal varied, depending on the composition of the medium. The values
of the emission anisotropy factor gem at 690 nm
for chloroplasts in the standard buffer varied from
90×10−4 to 170×10−4 depending on the sample. The
gem -value was 1.5–2.5 times higher than for lettuce
chloroplasts (∼60×10−4 by Gafni et al. 1975), but the
gem value of about 130×10−4 for lemon chloroplasts
(Gussakovsky and Shahak 1996) was in the same
range. There was almost no CPL signal of chloroplast suspended in the tricine buffer without sorbitol
(without osmotic pressure) and magnesium/potassium
(without ionic strength). In such a medium, macrostructures of the grana are absent and Photosystem
II (PS II) particles are destacked from each other
and mixed with Photosystem I (PS I) particles in the
membrane (Armond et al. 1977). As a reference,
we measured CPL of Chl in 80% acetone and found
no signal in accordance with data reported elsewhere
(Gafni et al. 1975). These findings indicate that individual Chls or Chls in pigment–protein complexes of
individual PS I or PS II particles do not exhibit a CPL
signal.
Figure 2. CD spectra of pea chloroplasts in the standard medium
(1; bold line), in 20 mM tricine–NaOH pH 7.6 supplemented with
0.4 M sorbitol and 5 mM MgCl2 (2; thin line), in 20 mM tricine–NaOH pH 7.6 supplemented with 0.4 M sorbitol (3, dashed
line), in 20 mM tricine–NaOH pH 7.6 supplemented with 0.4 M
sorbitol and 10 mM KCl (4, dotted line), in 20 mM tricine–NaOH
pH 7.6 without supplements (5, dashed and dotted line). Line (6,
bold dotted line) shows the absorption spectrum of chloroplasts in
the standard buffer.
We checked for possible artifacts occurring during
the measurement of CPL. Partially polarized excitation light can induce a component of linearly polarized
emission in the instrument with the 180◦ detection
scheme which can significantly influence the CPL signal (Steinberg and Gafni 1972; Riehl and Richardson
1986). We found no linearly polarized component
for chloroplast suspensions applying the approach of
doubling the modulation frequency (Steinberg and
Gafni 1972). Photoselection is known to produce an
additional lock-in amplifier output, mimicking CPL
(Steinberg 1978a). In our measurements, the 180◦
detection scheme and randomly oriented chloroplasts
in suspension did not produce this effect (Steinberg
1978a). In addition, the lock-in amplifier output signal
was not found in the samples of chloroplasts without
osmotic pressure and ionic strength, whereas it still
should give a signal if the photoselection would be
present.
Figure 2 shows the CD spectra (in the Qy absorption band) of pea chloroplasts in different media. Most
of the spectra (except the CD spectrum originating
from thylakoid membranes without granal ultrastructure) are dominated by large positive and/or negative
psi-type CD band (see also Garab et al. 1991; Barzda
et al. 1994). These spectra can be understood as a su-
87
Figure 3. Dependence of the negative 2min (panel A, filled circles)
and positive 2max (panel A, open circles) red CD bands as well
as the emission anisotropy factor, gem at 690 nm (panel B) for pea
chloroplasts on concentration of sorbitol in 20 mM tricine–NaOH,
pH 7.6. The straight lines represent the best fit obtained by the linear
regressions.
perposition of broad positive and negative CD bands
with the maxima of the bands shifted with respect to
each other (Finzi et al. 1989). We characterized the CD
spectrum by the intensities in the maximum (2max )
and the minimum (2min ), not by the intensities at specified wavelengths. The CD spectrum of thylakoids
without the granum ultrastructure consisted only of the
excitonic CD signal of the individual pigment-protein
complexes and did not contain the psi-type CD bands.
Effects of osmotic pressure, ionic strength and
photoinhibitory illumination
Ionic strength and osmotic pressure change the macroorganization of the grana in chloroplasts. These
changes affect the amplitude of the positive and negative psi-type CD band (Garab 1991, 1996; Barzda et
Figure 4. Dependences of the positive 2max (panel A, open
circles), negative 2min (panel A, filled circles) red CD bands and
the CD intensity at 436 nm (panel A, squares) as well as the emission anisotropy factor, gem at 690 nm (panel B) on the MgCl2
concentration for pea chloroplasts in 20 mM tricine–NaOH, pH 7.6
supplemented by 0.4 M sorbitol.
al. 1994). The osmotic pressure in pea chloroplasts
was varied using different concentrations of sorbitol
in the medium. The intensity of the negative CD band
linearly depends on the sorbitol concentration in the
0–0.4 M range, while the positive CD band has a tendency for saturation, stopping to increase at a sorbitol
concentration above 0.2 M (Figure 3A). The emission anisotropy factor gem at 690 nm is also linearly
dependent on the sorbitol concentration (Figure 3B).
Changes of the MgCl2 concentration in the chloroplast suspension containing 0.4 M sorbitol affected
both CD and CPL. Figure 4 shows that both the absolute value of the negative CD band intensity, |2min |
(panel A) and the gem -factor at 690 nm (panel B)
increase with the increase of the MgCl2 concentration from 0 to 0.6 mM and remain almost constant at
higher concentrations. The positive CD band, 2max
is approximately constant up to 0.6 mM MgCl2 and
88
Figure 5. Dependences of the positive 2max (open circles) and negative 2min (filled circles) red CD bands, as well as the emission
anisotropy factor, gem at 690 nm (rhombs) on the time of the photoinhibitory illumination of pea chloroplasts in the standard medium
supplemented with 5 mM DTT.
has a tendency to decrease at higher concentrations
(panel A). The saturation behavior of CD and CPL
suggests that at certain concentrations of sorbitol and
MgCl2 , maximal macro-organization of the grana can
be achieved.
At 10 mM KCl and 0.4 M sorbitol, the amplitude
of the positive CD band (Table 1) was similar to the
maximum value observed at 0.6 mM MgCl2 (Figure 4A). However, the intensity of the negative CD
band remains much smaller than the saturation value,
despite the fact that 10 mM KCl corresponds to a
higher ionic strength than 0.6 mM MgCl2 (see Figure
4 and data in Table 1). This illustrates that magnesium
and potassium ions act differently on the aggregation of pigment–protein complexes in the granum and
the aggregation does not depend linearly on the ionic
strength.
Changes in the ionic strength or osmotic pressure
of the medium also affect the photosynthetic activity of chloroplasts measured as Fv /Fm (Table 1). This
parameter correlates with the values of 2min and gem .
The macro-organization of the granum can also
be changed by prolonged photoinhibitory illumination (Gussakovsky et al. 1997). In the present study,
11 min of strong illumination of chloroplasts in the
standard medium supplemented by 5 mM DTT which
enhances the photoinhibitory effect of strong illumination (Demmig-Adams 1990; Thiele and Krause
Figure 6. The plots of the positive 2max (panel A) and negative
2min (panel B) red CD bands versus the emission anisotropy factor,
gem at 690 nm: open triangles and dashed-dotted line correspond to
the different concentrations of sorbitol as in Figure 3; filled circles
and solid line correspond to the different concentrations of MgCl2 as
in Figure 4; open circles and dotted line correspond to the different
times of the photoinhibitory illumination as in Figure 5; stars and
dashed line correspond to different compositions of the medium as
in Table 1. The straight lines represent the linear regressions, parameters of which are shown in Table 2. The bold line shows the linear
regression for all the data in the figure.
1994) significantly affects both the CPL and the red
CD signals (Table 1). Illumination of the DTT-treated
samples for different periods of time results in different levels of photoinhibition (determined by Fv /Fm )
and parallel decrease in the gem -factor and the intensity of the negative CD band (Figure 5). In contrast,
the positive CD band is not affected. Hence, for
DTT-stimulated photoinhibition, both 2min and gem
reflect a decrease in macro-organization of the grana
in chloroplasts in a similar manner as upon lowering
the concentration of sorbitol or magnesium ions.
89
Table 1. Intensities of the red CD bands and chlorophyll emission anisotropy factor gem of chloroplasts
in different media
Medium: 20 mM tricine
supplemented by 0.4 M
sorbitol and:
None
MgCl2
KCl
MgCl2 + KCl
MgCl2 + KCl + DTT
MgCl2 + KCl + DTT
Illumination
Fv /Fm
Positive band
2max , mdeg
Negative band
2min , mdeg
gem ×104
at 690 nm
No
No
No
No
No
11 min
0.493
0.733
0.479
0.743
0.695
0.073
31.8
57.8
59.1
55.8
36.5
49.4
–11.1
–54.4
–18.8
–55.1
–47.1
–30.0
34.2±1.5
134.1±2.8
53.7±3.4
138.6±2.6
133.0±2.6
63.3±1.4
The CD (Figure 2) and CPL data in each row were obtained for the same sample of chloroplasts. Chl
concentration of 20 µg/ml. Cuvette pathlength of 5 mM. The CPL data presented here and in Figure 1,
are obtained for the different isolations of chloroplasts. Concentrations of MgCl2 , KCl and DTT were
5 mM, 10 mM and 5 mM, respectively.
Table 2. Linear regression parameters
Plot
Correlation coefficient
Slope, degree
Intercept, mdegree
2max -vs-gem
From Table 1
Sorbitol variation
MgCl2 variation,
Photoinhibition
All the data
0.281
0.901
0.432
0.176
0.367
0.69
14.6
–1.36
0.57
1.35
42.0
11.5
74.1
45.4
42.3
2min -vs-gem
From Table 1
Sorbitol variation
MgCl2 variation
Photoinhibition
All the data
0.981
0.984
0.957
0.973
0.980
–3.90
–5.21
–3.67
–3.22
–3.76
0.12
1.96
0.52
–8.15
–0.51
The parameters of the linear regression were calculated according to Figure 6A (2max vs-gem ) and Figure 6B (2min -vs-gem ).
Correlation of CD and CPL
We compared the emission anisotropy factor, gem at
690 nm both with the negative 2min and positive 2max
CD bands for pea chloroplasts at different concentrations of sorbitol and cations and level of photoinhibition. Figure 6 shows the plots of the 2max (panel A)
and 2min (panel B) versus the gem -factor. The data fits
obtained by linear regression (lines in Figure 6: the
fitting parameters are presented in Table 2) show that
2min linearly correlates with the emission anisotropy
factor with a high correlation coefficient and an intercept near zero. The plot 2max versus gem (Figure 6A)
does not result in a linear correlation with a reasonable
correlation coefficient, and the intercept significantly
differs from zero (higher than the error of the 2 meas-
urement; Table 2). This also implies that 2max and
2min are not linearly correlated.
Discussion
Correlation of CPL and CD signals
Three independent factors (osmotic pressure, magnesium ions and photoinhibition), which affect the
macroorganization of the grana, influence the negative
CD band and the gem -factor at 690 nm to a similar
extent, resulting in a linear correlation between these
two quantities (Figure 6B, Table 2) which is reflected
by both a high value of the correlation coefficient and
an intercept close to zero (intercept was considered to
90
be close to zero if its value was similar to the error of
the CD or CPL measurement). The correlation parameters slightly differ when either sorbitol, magnesium
or photoinhibition affects the CD and CPL signals.
In contrast, the positive CD band is not linearly
correlated with gem and shows saturation behavior at
gem > 0.003. A possible explanation why the positive CD band intensity is not correlated with the
positive CPL signal, can be given if we consider that
the red-most positive CD band contains a large contribution of circular intensity differential scattering.
The psi-type CD bands are always a superposition of
the CD absorption and the CD scattering (Keller and
Bustamante 1986; Garab et al. 1988b). The CD scattering has a strong wavelength dependence caused by
an increase of the differential scattering towards the
edges of the absorption bands and beyond and is most
clearly expressed in the CD spectrum as ‘long tails’
outside the red wings of the principle absorption band
(Bustamante et al. 1983, Garab et al. 1988b). Significant contributions of the CD scattering can also be
found inside the CD absorption band with the maximal relative contribution shifted towards the red side
of the principal absorption band (Bustamante et al.
1983; Keller and Bustamante 1986). So, one might
expect that the positive CD band intensity would also
correlate with the CPL signal if the CD scattering
contribution would be corrected for.
The psi-type CD absorption spectrum of chloroplasts can be a superposition of the positive and
negative CD bands with maxima shifted with respect
to each other because differential polarization imaging showed the separated positive and negative CD
bands which originate from different locations of the
same chloroplast (Finzi et al. 1989). As CD and CPL
are complementary phenomena, the CPL spectrum of
chloroplasts is expected to be split and to contain positive and negative bands. It is not clear to us why a
negative CPL band is not observed.
Origin of CPL of pea chloroplast
In most cases, the CPL signal originates from the
asymmetry or chirality of an emitting molecule. In a
similar way, the chiral molecule will give rise to a CD
signal due to preferential absorption of left or right
circularly polarized light (see comments in ‘Materials and methods’ on camphorquinone). When a chiral
molecule is excited by left or right circularly polarized
light, the respective emission is also left or right circularly polarized. This is a trivial chiroptical effect. Is
it possible that the CPL signal, which depends on the
macrostructure of the granum, is caused by such trivial
chiroptical effect? The following facts indicate that
this is not the case. The positive gem -factor correlates
with the negative CD band while the sign must be the
same in the case of a trivial chiroptical effect. The CD
signal at the Chl fluorescence excitation (436 nm) did
not depend on the MgCl2 concentration (Figure 4) but
the CPL signal was varied significantly, while both CD
and CPL signals must vary similarly because of the
trivial chiroptical effect. The gem -factor was sensitive
to the macrostructure of chloroplasts and appeared to
be more than one order of magnitude larger in the intact grana in comparison with the unstacked thylakoid
membranes while the trivial chiroptical effect should
not depend on the macrostructure of the grana. It is
very unlikely that stacking of the grana would deform
the planar chlorophyll molecules to such a large extent
that it would explain the enormous CPL signal.
In a previous study (Garab et al. 1991; Barzda et al.
1994), the CD signals of chloroplasts, which depend
on the macroorganization of the grana, were attributed to a psi-type origin and they, for instance, are
much larger than those of the major light-harvesting
complex of green plants (Nussberger et al. 1994).
Although the correlation between the CD and CPL
signals can be explained only with some assumptions
which have to be tested, the sensitivity of CPL to the
changes in macrostructure is evident. On this basis, we
tentatively assign the macro-organization-dependent
CPL to a psi-type origin. The psi-type CD signals
were observed in viruses, chromosomes and other
DNA aggregates (for review see Tinoco et al. 1987).
The psi-type CD theory (Keller and Bustamante 1986)
pointed out that an individual chromophore is influenced by the electric fields of all other chromophores
in the chiral aggregate, and the absorption has to be
considered as a collective property of all coupled chromophores rather than as a property of an individual
pigment. The latter is revealed in the CPL signal where
the emission by a single chromophore can be influenced by the electric field of all other chromophores
in the chiral aggregate which give rise to a large gem
factor.
Concluding notes
It was shown that the CPL technique can be used to
study changes in the macro-organization of photosynthetic complexes. This offers the possibility to use
CPL as a non-invasive spectroscopic tool for struc-
91
tural analysis in the samples of high optical density
when the use of circular dichroism is impossible (for
example, granum ultrastructure in green leaves as a
function of changes in the environment). The obtained
results open the pathway for future studies on the longrange packing of photosynthetic systems in individual
chloroplasts under a large variety of conditions with
the use of CPL-based microscopy.
Acknowledgements
Dr E. Haas, Bar Ilan University, is acknowledged
for enabling the use of the CD and CPL instruments,
which were supported in part by the Israel Science
Foundation founded by the Israel Academy of Sciences and Humanities. The authors thank to Dr R.
Van Grondelle and Dr G. Garab for helpful suggestions and discussion. The research was supported in
part The United States-Israel Binational Agricultural
Research and Development Fund (BARD), Grant #IS2710-96 to YS. VB was supported by an EMBO
long-term fellowship and travel grant of the European
Science Foundation, Programme Biophysics of Photosynthesis.
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