全 文 ：Received 30 Jun. 2003 Accepted 20 Oct. 2003
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 423-427
A Circular Dichroism Spectroscopic Study Revealing the Cause of the
Changes of Chlorophyll a Fluorescence Induction of
Photosystem Ⅱ During Heat Treatment
LI Dong-Hai1, RUAN Xiang1, XU Qiang1, GONG Yan-Dao1, ZHANG Xiu-Fang1, ZHAO Nan-Ming1*
WANG Ke-Bin2, KUANG Ting-Yun2
(1. State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and
Biotechnology, Tsinghua University, Beijing 100084, China;
2. Photosynthesis Research Center, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China)
Abstract : Chlorophyll a fluorescence and circular dichroism (CD) spectra of photosystem Ⅱ (PSⅡ)
membrane were measured after heat treatment. The chlorophyll fluorescence parameter Fo remained
stable after treatment at the temperatures from 30 ℃ to 40 ℃ and then reached a maximum after
treatment at 55 ℃. In PSⅡ membranes and LHCⅡ (light-harvesting chlorophyll a/b binding complex)-
enriched complexes, anomalous CD signals with extremely large amplitudes occurred during the heat
treatment. The temperature corresponding to the maximum anomalous CD intensity peaking at 677 nm
was 40 ℃. The results indicate that the aggregation state of the LHCⅡ in PSⅡ is related to the anomalous
CD signal, and can be an important factor influencing Fo in the heat treatment of PSⅡ membrane.
Key words: chlorophyll; circular dichroism (CD); photosystem Ⅱ (PSⅡ); light-harvesting chlorophyll
a/b binding complex (LHCⅡ)
Circular dichroism (CD) spectroscopy is a powerful and
noninvasive technique to obtain structural information in
samples of biological origin. In molecular complexes or small
aggregates, CD signal is generally induced by short-range
interact ions , e.g ., due to excitonic interact ions between
molecules on neighboring particles, and a distortion by
differential scattering. However, the large aggregation-in-
duced CD could not be explained by above theory. Theory
predicts that in polymer- and salt-induced aggregates the
magnitude of the anomalous CD, at a constant density of
chromophores and a constant pitch o f the macro-helix, is
cont rolled by the volume of the aggregate (Keller et a l.,
1986). Barzda et al. (1994) have investigated the CD of pig-
ment-protein complexes in different aggregation states both
in thylakoid membranes and in isolated LHCII, and p ro-
vided direct experimental ev idence that the magnitude of
the major CD bands increases with the s ize o f the
aggregates. In their experiments, they treated the materials
with MgCl2 and Triton X-100 in order to cause the anoma-
lous CD with extremely large amplitudes. Then, are there
other ways that can also induce the anomalous CD? In
photosynthetic systems, what causes the anomalous CD?
Chlorophyll (Chl) a fluorescence is a useful and routine
probe for in fo rmat ion on the various aspects o f
photosynthesis, and a good non-destructive technique for
evaluating the heat-induced effects on photosystem Ⅱ (PS
Ⅱ) photochemical activities (Schreiber et al., 1977; Berry
et al., 1980). For the plant leaf and chloroplast, the effects
of heat treatment on the Chl a fluorescence parameters have
been studied by many researchers, and an increase of Fo
and decreas e of bo th Fv and Fv/Fm have been reported
(Yamane et a l., 1997; Yamane et a l., 1998; Pospíšl et al .,
1998). Mechanisms about changes of the chlorophyll a fluo-
rescence parameters have also been suggested and testified.
So far, some reports have shown that the increase in the Fo
level by heat can be attributed to the dissociation of light-
harvesting chlorophyll a/b binding complex (LHCⅡ) from
PSⅡ core complex (Schreiber et al., 1978) and inhibition of
electron flow from QA to QB (Bukhov et al., 1990). However,
the cause of the little change of the Fo level at lower heat
level (30-40 ℃) has not been well explained.
In the present experiment, PSⅡ membrane was adopted
to study the heat-induced changes of CD and chlorophyll
a fluorescence. The results indicated that during the heat
treatment of PSⅡ membrane anomalous CD signals was
strongly related to the aggregation state of LHCⅡ which
could be a factor influencing the chlorophyll fluorescence
parameter Fo.


Acta Botanica Sinica 植物学报 Vol.46 No.4 2004426
in s perm heads , condens ed chromatins , and DNA
aggregates, respectively. In general thylakoid membranes
and in macroaggregates of LHC Ⅱ, nonconservative CD
signals with extremely large amplitudes have been observed.
In the present experiment, in PSⅡ membranes, anomalous
CD s ignal with extremely large amplitudes also occurred
during the heat treatment. The anomalous increase of CD
signal could no t be due to the increase of short-range
interaction, e.g. excitonic interactions between chlorophyll
molecules, and a distortion by differential scattering (Garab
et al., 1988). Such anomalous CD may be associated with
the state of aggregation of the PSⅡ complexes (Gregory et
al., 1980), and at tributed to long-range interactions of ag-
gregation (Simidjiev et al., 1997). According to the results
shown in Fig. 2, the anomalous CD signals were not in-
duced by the state of aggregation of the PSⅡ membrane,
but that of the LHCⅡ during the heat treatment. Barzda et
al. (1994) also observed the Triton X-100-treated anoma-
lous CD of LHCⅡ and provided direct experimental evi-
dence of the size dependency of CD in macroaggregates.
The results indicated that though the treatments were dif-
ferent in these experiments, the mechanism of the changes
of LHCⅡ cou ld be accordant. In PSⅡ membranes and
LHCⅡ -enriched complexes , the anomalous CD may be
involved in the changes of the aggregation state of LHC
Ⅱ.
During the heat treatment, the increase of Fo with the
rise of temperature was usually attributed to detachment of
LHCⅡ from PSⅡ co re complex (Schreiber et a l., 1978)
and/or inhibition of electron flow from QA to QB (Bukhov et
al., 1990). Since Fo maintained stable for the temperature
range from 30 ℃ to 40 ℃ in the present experiment (Fig. 1),
some other factors migh t exist to influence Fo value. Fig.
2a shows that the CD signal rapidly increased at the tem-
peratures from 30 ℃ to 40 ℃, indicating that the changes
of the aggregation state of LCHⅡ occurred, in other words,
the size of aggregation of LHCⅡ in the heat-treated PSⅡ
could be bigger than that in the native LHCⅡ (Barzda et
al., 1994; Simidjiev et al., 1997). The increase of the aggre-
gat ion of LHCⅡ could quench its chlorophyll fluores-
cence (Mullet et a l., 1980; Ruban et a l., 1992). In view of
such decreasing effect on fluorescence, the stableness of
Fo at the temperatures from 30 ℃ to 40 ℃ is reas onable.
Figure 2a also shows the reduction o f CD intensity after
treatment at temperatu res from 40 ℃ to 55 ℃, indicat ing
the size of aggregation of LHCⅡ became smaller, which
would increase the fluorescence Fo value. This is consis-
tent with the results shown in Fig.1. Therefore, our results
showed that the s tate of aggregation o f LHCⅡ can be a
factor influencing Fo in the heat-induced denaturation of
PSⅡ. During the heat treatment of PSⅡ membrane for the
temperature range from 40 ℃ to 55 ℃, the decrease in the
excitonic interaction between chlorophyll molecules may
also be involved in the change of the CD signals.
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(Managing editor: HE Ping)