Full text of DOI:10.1007/BF02879811

Vol. 44 No. 4
SCIENCE IN CHINA (Series B)
August 2001
Ultrafast spectroscopy studies on the mechanism of electron
transfer and energy conversion in the isolated pseudo
ginseng, water hyacinth and spinach chloroplasts
XU Sichuan (徐四川)1, SUN Zhaoyong (孙照勇)1, AI Xicheng (艾希成)1,
FENG Juan (冯 娟)1, ZHANG Qiyuan (张启元)1, ZHANG Xingkang (张兴康)1,
YU Fei (郁 飞)2, TANG Chongqin (唐崇钦)2, LI Liangbi (李良璧)2
& KUANG Tingyun (匡廷云)2
1. State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center of Molecular Science, Insti-
tute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China;
2. Photosynthesis Research Center, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Correspondence should be addressed to Xu Sichuan(email: xusc@chem.pku.edu.cn)
Received February 20, 2001
Abstract The spectroscopy characteristics and the fluorescence lifetime for the chloroplasts iso-
lated from the pseudo ginseng, water hyacinth and spinach plant leaves have been studied by
absorption spectra, low temperature steady-state fluorescence spectroscopy and single photon
counting measurement under the same conditions and by the same methods. The similarity of the
absorption spectra for the chloroplasts at room temperature suggests that different plants can effi-
ciently absorb light of the same wavelength. The fluorescence decays in PS II measured at the
natural QA state for the chloroplasts have been fitted by a three-exponential kinetic model. The
three fluorescence lifetimes are 30, 274 and 805 ps for the pseudo ginseng chloroplast; 138, 521
and 1494 ps for the water hyacinth chloroplast; 197, 465 and 1459 ps for the spinach chloroplast,
respectively. The slow lifetime fluorescence component is assigned to a collection of associated
light harvesting Chl a/b proteins, the fast lifetime component to the reaction center of PS II and the
middle lifetime component to the delay fluorescence of recombination of P6+80 and Pheo−. The ex-
citation energy conversion efficiency (η) in PS II RC is defined and calculated on the basis of the
20 ps electron transfer time constant model, 60%, 87% and 91% for the pseudo ginseng, water
hyacinth and spinach chloroplasts, respectively. This interesting result is in unconformity with what
is assumed to be 100% efficiency in PS II RC. Our result in this work stands in line with the 20 ps
electron transfer time constant in PS II rather sound and the water hyacinth plant grows slower
than the spinach plant does as envisaged on the efficiency. But, our results predict that those
plants can perform highly efficient transfer of photo-excitation energy from the light-harvesting
pigment system to the reaction center (closely to 100%). The conclusion contained in this paper
reveals the plant growth characteristics expressed in the primary processes of photosynthesis and
a relationship between a plant growing rate and its spectroscopy characteristics and fluorescence
lifetimes, namely, the slower a plant grows, the less excitation energy conversation efficiency used
might be anticipated.
Keywords: pseudo ginseng, water hyacinth, chloroplast, single photon counting, fluorescence lifetime, excitation energy
conversation efficiency.
In 1960, Arnold and Clayton[1] reported that the first light-chemical step in the purple bacte-

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ELECTRON & ENERGY TRANSFER IN CHLOROPLASTS
367
rium photosynthesis is the electric charge separation called primary reaction, which results in
holding dynamics for photosynthesis. Currently, the primary processes of photosynthesis in green
—
plants have still been an important topic of interest[2 5]. Chlorophyll (Chl) fluorescence has been
used to understand excitation energy transfer processes, pigment orientation and protein organiza-
—
tion in extensive experimental and theoretical work for almost two decades[6 9]. It is well known
that the primary processes of photosynthesis in green plants have been shown to be highly effi-
cient and energy losses constitute only minor deactivation pathways. But, there is still a finite
probability that it will decay either by non-radiative process or by fluorescence before trapping
can take place during excitation migration in the antenna, the separation and stabilization[9].
Green plant growth is related with or controlled by a number of factors. One of them is plant
genome, an intrinsic nature that dominates green plant growth. Recently, it was reported that the
genome sequences of a number of bacterial species and several eukaryotes were completed while
nucleotide sequencing of the Arabidopsis genome is nearing completion and sequencing of the
rice genome has been just finished, and a large amount of expressed sequence tag information is
being obtained for many other plants[10,11]. This wealth of sequence information is used to acceler-
ate progress toward a comprehensive understanding of the genetic mechanisms that control plant
growth, development and response to the environment. Although different plant growths and de-
velopments are dominated by their genomes which determine how the plants use the energy,
however, according to the law of energy conservation, the faster a plant grows, the more the en-
ergy should be preserved, and the slower a plant grows, the less the chemical energy should be
stored. It depends on the efficiency of absorbing light turned to excitation energy by chlorophyll
and carotene in per mass chloroplast, the translation energy efficiency and the excitation energy
conversation efficiency. In other words, the difference may be expressed in the primary processes
of photosynthesis.
Based on the analysis above, we adopt a few of different plant chloroplasts and compare
them with their primary processes of photosynthesis to obtain more information so that the char-
acteristics of the primary processes of photosynthesis in different green plants can be further
understood. In this work, three kinds of chloroplasts isolated from pseudo ginseng (Panax
pseudo-ginseng)[12] growing up for 3—7 a at Wenshan of Yunnan Province, and water hyacinth
(Eichhornia crassipes)[13] growing rapidly at Dian Chi in Kunming of China and Spinach
(Spinacia oleracea L.) purchased from local market, which are internationally extensively studied
by researchers in the chemical, biological and physical fields, have been studied by absorption
spectra, low temperature steady-state fluorescence spectroscopy and single photon measurement
under the same conditions. We will distinguish and argue on the origin of fluorescence decay
component, define and measure the excitation energy conversation efficiency in the primary proc-
esses of photosynthesis to probe the electron transfer time constant and the growth characteristics
of green plant interrelated with its spectroscopy characteristics and fluorescence lifetimes.

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1
Materials and methods
1.1 Isolation of chloroplast
The isolation method for the chloroplasts can be seen in refs. [14, 15]. Deveined fresh leaves
washed by distilled water were homogenized by a mixer in a preparation medium buffer (pH 7.4)
containing 0.2 mol/L NaCl, 0.1 mol/L sucrose, and 0.05 mol/L phosphate. After filtration through
4 and 16 layers of gauze, the green juice was centrifuged at 600 g for 5 min and the precipitate
was discarded. The supernatant was again centrifuged at 1500 g for 10 min and the precipitate was
suspended in a medium buffer (pH 6.9) containing 0.05 mol/L NaCl, 0.3 mol/L sucrose, and 0.05
mol/L phosphate. Preparation procedures for the chloroplasts were finished at 4℃ and in dark.
The isolated samples were stored in liquid nitrogen until use.
1.2 Experimental methods
Chlorophyll protein content and Chl a/b ratio for the chloroplasts were determined according
to the method in ref. [16] on UV-190 split-beam spectrophotometer. The result is listed in table 1.
Among the three chloroplasts, the value of Chl a/b ratio is the biggest in the water hyacinth
chloroplast and the smallest in the pseudo ginseng chloroplast, meaning that in the water hyacinth
chloroplast the amount of Chl b is relatively less than Chl a, and vice versa in the pseudo ginseng
chloroplast, compared with that occurring in the water hyacinth and spinach chloroplasts.
Table 1 The Chl a / b ratio for the pseudo ginseng, water hyacinth and spinach chloroplasts
Water hyacinth
3.36
Spinach
3.03
Pseudo ginseng
2.43
Chl a/b ratio
Absorption spectra and their fourth derivative absorption spectra were recorded by
UVIKON943 double-split-beam spectrophotometer under the condition of 4℃ and avoiding
light[17]. Low temperature steady-state fluorescence spectroscopies were measured according to
the method of Tang et al.[14] at liquid nitrogen temperature using Hitach Ltd. F-4500 fluorescence
split-beam spectrophotometer. The Chl concentrations of the samples were diluted to 10 μg/mL.
Time-correlated single photon count is an experimental method to measure fluorescence de-
cay time[18,19] with the advantages of high sensitivity, wide spectrum and weak light strength to
radiate samples[20]. Time-resolved fluorescence measurements were performed using a synchro-
nously pumped and cavity-dumped dye laser system (Spectra Physics: SP3500 Model) with a
mode-locked YAG laser (Spectra Physics: SP3800 Model) as the pumping source. An intracavity
acousto-optically dumped Rhodamine 6G dye laser at 570—610 nm for pulse selecting at 800 kHz,
was used. The maximum laser pulse intensity (1×107 photons per cm2) was attenuated with output
laser power of 50 mW. A microchannel plate detector (Hamamatsu, Model R1645-01u) was ap-
plied to detecting single photon. The 10 mV anode signal from the detector was amplified with a
Hewlett Packard HP8447f pre-amplifier, with amplifier combination being 0.6—1.0 V. Fluores-

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ELECTRON & ENERGY TRANSFER IN CHLOROPLASTS
369
cence was collected at 90° using a Ditric Optics 3-plate birefringement. The instrument response
function had an FWHM of 100 ps. Total 1024 channels of the single photon with 25 ps per chan-
nel were recorded. The count is 10000 at peak. This single-counting system detects light pulses of
typical 30 ps duration at variable repetition rates. The chloroplast samples all were excited at 580
nm and detected at 682 nm. The optical length of the cuvette for the measurement was 1.0 cm.
The chloroplast suspension was diluted with the buffer to a final concentration of 10 μg/mL Chl,
0.4 mol/L sucrose, pH: 6.5, 50 mmol/L Mes, and 10 mmol/L NaCl. Life-times according to mul-
tiexponential kinetics were calculated by an iterative deconvolution method using a semi-linear
Marqurdt-algorithm. The quality of the fits was judged by both a reduced X2 criterion and a plot of
weighted residuals. The fitting program (ASUFIT) used in this work was presented by Photosyn-
thesis Research Center in Arizona State University.
2
Experimental results
2.1 UV absorption spectra
Fig. 1 (a) shows absorption spectra of the pseudo ginseng, water hyacinth and spinach
chloroplasts, and fig. 1(b) is their fourth derivative absorption spectra at room temperature. Ab-
sorption peaks at 680 and 438 nm and shoulder peaks at 649 and 484 nm are generated by Chl a
and Chl b absorbing light, respectively. Besides, carotenoid originates absorption peak at 438 nm.
In the fourth derivative absorption spectra, absorption peak at 680 nm is split into two peaks at
669 and 682 nm, responsible for two different states of Chl a molecules. Absorption spectra and
their fourth derivative absorption spectra both display an analogy for the pseudo ginseng, water
hyacinth and spinach chloroplasts efficiently absorbing light of the same wavelength. However,
fig. 1 shows that the absorption intensity at 400—500 nm for the water hyacinth chloroplast is
stronger than those for the other chloroplasts. This result discovers that the same preparation ap-
Fig. 1. Absorption spectra at room temperature for the pseudo ginseng, water hyacinth and spinach chloroplasts.
(a) Absorption spectra; (b) the fourth derivative absorption spectra.

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pears to remove an amount of carotenoid molecules less for the water hyacinth chloroplast clued
further by the strongest absorption peak at 438 nm.
2.2 Fluorescence spectra at low temperature
Fig. 2 shows fluorescence emission spectra of the pseudo ginseng, water hyacinth and spin-
ach chloroplasts at liquid nitrogen temperature. In the fluorescence emission spectra derived from
both Chl a absorbing at 436 nm (λex = 436 nm) and Chl b absorbing at 480 nm (λex = 480 nm),
fluorescence emission at 683 nm comes from PS II and 731 or 738 nm from PS I[21]. Only for the
spinach chloroplast, can the fluorescence emission peak at 691 nm be generally reckoned to come
from its PS II internal antenna. With respect to the fluorescence emission for the spinach chloro-
plast, the fluorescence emission in PS I for the pseudo ginseng chloroplast exhibits red shift by 10
nm. In the PS II reaction center, the electric charge separation makes oxidized and reduced species
with highly energy appear. In the side of oxidized species, water is oxidized into oxygen and the
reduced species connects with the PS I that possesses its own light-harvesting pigment system. In
the primary processes of photosynthesis for green plant photosynthesis system, to maintain highly
efficient photosynthesis rate, it is necessary to keep balance between two photo systems that photo
quantum nearly equally enters into PS II and PS I[22,23]. Low temperature steady-state fluorescence
spectroscopy of PS II and I may account for the balance at some extent. If F683, F731 and F738 rep-
resent PS II and PS I relative fluorescence intensity for the three plant chloroplasts, the value of
F731 / F683 (spinach), F732 / F682 (water hyacinth) and F738 / F683 (pseudo ginseng) (see fig. 3 (a))
can be applied to judging the distribution of excitation energy between two photo systems and
estimate the balance. The values of ratios between two photo systems are 0.29，0.37 and 0.85 for
the pseudo ginseng, water hyacinth and spinach chloroplasts, respectively, which discloses that the
balance for the spinach chloroplast is the best, while the balance for the pseudo ginseng chloro-
plast the worst. The similar values are 0.31, 0.49 and 0.90 for the pseudo ginseng, water hyacinth
and spinach chloroplasts observed in fig. 3 (b) by Chl b absorbing at 480 nm. It is interesting to
notice that pseudo ginseng plant growing slower is in accordance with the less balance of distribu-
tion of excitation energy between two photo systems. This result may not be an accidental coinci-
dence, possibly an indication for the less efficient photosynthesis in the pseudo ginseng chloro-
plast.
The fluorescence excitation spectra of the pseudo ginseng, water hyacinth and spinach
chloroplasts at 77 K are shown in fig 3. An observation is that in fig. 3 (a) the same intensity peak
at 437 nm arising from Chl a as that at 483 nm from Chl b appears in the pseudo ginseng chloro-
plast. This result illustrates that light energy absorbed by Chl a and b in the pseudo ginseng
chloroplast fabricates a similar contribution to fluorescence emission (F683) in PS II. In fig. 3 (b),
the less ratio of contribution to PS I fluorescence emission (F740) by Chl b absorbing at 474 nm is
ascertained.

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Fig. 2. Fluorescence emission spectra for the pseudo ginseng, water hyacinth and spinach chloroplasts at 77 K.
Fig. 3. Fluorescence excitation spectra for the pseudo ginseng, water hyacinth and spinach chloroplasts at 77 K.
2.3 Fluorescence lifetimes
During the primary processes of photosynthesis, the non-radiative process and the radiative
process may both play an important role in the decay of excitation energy in the plant. In this work,
we have taken fluorescence decay lifetimes including the two processes measured by a
time-resolved chlorophyll fluorescence of the isolated samples.
The measured fluorescence decay curves in PS II at room temperature for the pseudo ginseng,
water hyacinth and spinach chloroplasts are shown in fig. 4. At this point, we do not add DCMU

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or potassium ferricyanide into the samples to
affect the QA state in order to study the near
natural states in PS II of the chloroplasts for a
comparison. Furthermore, we try to maintain the
same experimental conditions in all stages for
the different chloroplasts including Chl a/b ratio
determined, absorption spectra recorded, low
temperature steady-state fluorescence spectros-
copy measured and time-resolved fluorescence
measurements performed. It is found that the
quality of the fits judged by both a reduced χ2
criterion and a plot of weighted residuals was not
improved from three kinetic components to four
kinetic components so that a three-component
model to fit fluorescence decay curves was used.
Fig. 4. Instrument response curve and fluorescence decay
curves in PS II at room temperature for 1, water hyacinth; 2,
spinach; 3, pseudo ginseng; and 4, laser.
The lifetime of slow component depends on the fitting range. In table 2, the results for a fitting
range of 4.0, 4.5 and 5.0 ns are listed including their values of a reduced χ2 criterion less than 1.5
and amplitude values. The weighted residuals are given in fig. 5. Their values responsible for the
accuracy of a fitted result are all less than ± 4 in the fitting range with a uniform line.
The fitted results illustrate that among
the three chloroplasts for the pseudo ginseng
chloroplast the fluorescence lifetimes of three
decay components in PS II are the smallest.
The lifetime (τ f) of fast fluorescence decay
component in the PS II of the pseudo ginseng
chloroplast is just 30 ps with the largest am-
plitude 60%. In the spinach chloroplast the
largest amplitude is the middle lifetime com-
ponent (66%, 465 ps), but in the pseudo gin-
seng chloroplast the largest amplitude is the
fast lifetime component (60%, 30 ps), while
the lifetime of fast component is 190 ps for
the spinach chloroplast. It generally agrees
with the origin of fluorescence decay for the
slow component arising from the peripheral
antenna so that it may account for that the
peripheral antenna little decays absorbed
Fig. 5. The weighted residuals (all less than ±4) for the fitting
ranges of 4.0, 4.5 and 5.0 ns for the pseudo ginseng, water hya-
cinth and spinach chloroplasts, Y-coordinate is for the weighted
residuals and X-coordinate for the fitting ranges. 1, Water hya-
cinth; 2, spinach; 3, pseudo ginseng; 4, laser.

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373
light energy during the primary processes of photosynthesis in green plants, because of the ampli-
tude of the slow lifetime component being the smallest for all the chloroplasts and less contribu-
tion to fluorescence decay. It should be mentioned that the lifetime of fast component is obviously
distinct, lowered to 30 ps for the pseudo ginseng chloroplast corresponding to 200 and 140 ps for
the water hyacinth and spinach chloroplasts while the amplitude highlighted from 30%—40% to
60%. Detailed discussion for the origin of fluorescence component and applications will be given
in the next section.
Table 2 The fitted results of fluorescence decay curves of PS II for the pseudo ginseng, water hyacinth
and spinach chloroplasts
0—4 ns
0—4.5 ns
0—5 ns
A (%)
30
66
4
χ2
A (%)
28
68
4
χ2
A (%)
32
64
4
χ2
τf /ps
196
465
1469
135
504
1437
29
τf /ps
190
455
1378
138
515
1476
30
τf /ps
205
477
1530
143
533
1569
31
Spinach
1.05
1.03
1.05
42
50
8
43
50
7
44
49
7
Water
hyacinth
1.11
1.30
1.03
1.42
1.03
1.54
59
38
3
60
38
3
60
38
2
Pseudo
ginseng
265
731
274
801
282
883
3
Discussion
Explanation of fluorescence original for the experimentally observed complex decay kinetics
in a green plant chloroplast has been a subject of intense discussion. Two models were found to be
kinetically appropriate in isolated intact PS II of chloroplast of green plant. The simplest model in
a rigorously defined quantitative manner, which has been used to interpret steady state fluores-
cence measurements, is the bipartite model[24] which involves two species, antenna chlorophyll
and a reaction center, implying that the light harvesting chlorophyll a/b protein and PS II core
antennae are so tightly coupled that they may be considered as one species. In this model, it ig-
nores all details of energy transport through the antenna and treats only the coupling between an-
tenna A and reaction center (RC or P680). The reversible radical pair model[5,6,25] is based on fast
(few ps) equilibration of exciton distribution between all PS II Chl and only considers the rate
constants of electron transfer reactions in the reaction center[26] that is the limiting step in charge
separation. The tripartite model and heterogeneous bipartite dynamics model were put up by
modifying the bipartite model[24]. By changing PS II RC to closed state, or open state, or any state
between them, more information can be obtained to understand the kinetics and energetics of the
exciton trapping, charge separation, charge recombination, and charge stabilization processes in
PS II and some of experimental measured fluorescence decays attach a description of components
from PS I or PS II α/PS II β heterogeneous units[5,25] brought forward throughout different states
of Chls conjoint to the reaction center in PS II with their own features of the distinct energy trans-

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Vol. 44
fer rates. However, our recent experiment can argue that fluorescence decay components of fast
and middle lifetimes in PS II are independent of PS I[27]. The concept of α and β centers in terms
of PS II centers of different antenna sizes located in different areas of the thylakoid membrane has
recently been questioned and therefore such an interpretation shows a certain degree of uncertain-
ty[9,28,29]
.
Fr□e Chl in solution has a typical lifetime around 5.0 ns[30]. In isolated LHCII the lifetime
drops to 1.2 ns due to increased non-radiative decay[8]. Although there exist the controversies on
the three-component or four-component theoretical models and the argument on the origin of fluo-
rescence component in the green plant chloroplast, it generally agrees with the assignment of the
slow lifetime component to a pool of free chlorophyll[6,31], as suggested by Marder and Raskin[32]
,
which is supported by our measurements of relative variable fluorescence in PS II from the
chloroplasts due to different states of chlorophyll[33]. However, the assignment of the fast and
middle components is still in conflict with each other. Schatz et al.[5] reported a conclusion that at
least two pigment pools contribute to the fast component. Recently, it is reported that the explana-
tions of the reversible radical pair model are most compatible with recent experimental data on
charge separation time constants[26]. The reversible radical pair model may predict that the fluo-
rescence decay of any PS II population in the chloroplasts will be composed of two exponential
components in that these systems in this paper have the characteristics of the fast and middle
components. Mainly based on the two models and our experimental results, we suggest a hypothe-
sis for the assignment of the fast and middle lifetime components, i.e., the fast lifetime component
originates from the reaction center of PS II, and the middle lifetime component may be consistent
+
with the delay fluorescence of recombination of P680 and Pheo−. After the slow lifetime compo-
nent has been attributed to a collection of associated light harvesting Chl a/b proteins, for the
largely releasing absorbed light energy due to less efficient photosynthesis in the pseudo ginseng
chloroplast, it is an appropriate suggestion that the fast lifetime component is assigned to the reac-
tion center in PS II which is in a broad sense compatible with the result of Roelofs et al.[28], that is,
the α-β heterogeneity of PS II results in two pairs of lifetimes for fast and middle compo-
nents[28,29]. In PS II, the fluorescence emission peak at 691 nm of the spinach chloroplast is gener-
+
ally believed to come from its PS II internal antenna. Due to the reaction of recombination of P680
and Pheo−, the middle fluorescence lifetime is delayed by more than 200 ps compared with the
fast fluorescence lifetime[32]. Our experimental results tally with the conclusion that there exists
the delayed 200 ps fluorescence lifetime. Our assignment of the fast lifetime component attributed
from the reaction center of PS II, the middle lifetime component from the delayed fluorescence of
+
recombination of P680 and Pheo− and the slow lifetime component from a c llection of associ-
ated light harvesting Chl a/b proteins not only coincides with the charge separation controlled
model in PS II reaction center but also is able to interpret the current experimental observations
very well.

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375
For the fast lifetime component, its fluorescence kinetics can be described as follows:
P
680 + hν → 1P680* (or 1A * + P680 → A + 1P680*)
Rate: Ia (ka [1A *] [P680]),
[1P680*] kf ,
1P680* → P680 + hν
1P680* → P680
[1P680*] kic,
[1P680*] kisc,
[1P680*] kr,
1P680* → 3P680
*
+
1P680* → P680
In this model, Ia (or ka ) represents absorbed photo quantum or the transfer rate constant from
any antenna chlorophyll to the P680, kf is the radiative rate constant for fluorescence emission, kic
and kisc represent nonradiative decay rate constant for interconversion and intersystem crossing,
respectively, and kr is charge separation rate constant. Notice that in this model the reversible en-
ergy transfer from the P680 to other chlorophylls and the delay fluorescence from the recombina-
tion of P680+ and Pheo− were not considered simply.
According to Cowan and Drisko[34] (assumed just by absorbing photo quantum to excite P680
for simplifying the equation), we can obtain
d[1P680*]/dt = 0 = Ia − [1P680*]kf − [1P680*]kic − [1P680*]kisc − [1P680*]kr,
then
[1P680*] = Ia / (kf + kic + kisc + kr),
next
Фf = kf / (kf + kic + kisc + kr)
and
τ f = 1 / (kf + kic + kisc + kr),
where τ f is experimental fluorescence lifetime which is controlled by kf, kic, kisc and kr, etc. When
DCMU or potassium ferricyanide is added into the samples of the chloroplasts, the QA state of PS
II changes, resulting in the alternation of the values of kf, the values of kic, kisc and kr are increased
or dropped. Therefore, it has brought about difficulty in analyzing the fluorescence component
origin. In this work, it was emphasized that the fluorescence decay curves of PS II were measured
at the same condition of the natural states in PS II.
It is still an unsolved controversy for the charge-separated rate constant of PS II reaction cen-
ter. Two models 20 and 3 ps charge-separated rate constant to explain the controversy have been
—
put forward[26,35 45]. Klug and his coworkers had published a series of papers in support of a ca.
20 ps effective time constant for the appearance of the charge-separated state. The conclusion was
supported by the experiments reported by McCauley et al. and Freiberg et al. This effective time
constant is consistent with a trap-limited model for charge separation in PS II. On the other hand,
several groups had published data interpreted as supporting a ca. 3 ps time constant for the
—
primary charge separation[41 45]. A 3 ps time constant is consistent with low-temperature
hole-burning experiments and early predictions based on kinetic models and picosecond fluores-
cence experiments[5] and also in agreement with the well-established 2.5—3.5 ps time constant for

376
SCIENCE IN CHINA (Series B)
Vol. 44
primary charge separation in the reaction center of purple bacteria[24]. Donovan et al.[46] tried to
argue this controversy. They found the sensitivity of the observed transient absorption signals de-
pending on the laser intensity. A variety of fast components, unobserved at low laser intensities,
become prominent at higher laser intensities. At low excitation energy the signals observed are
most consistent with a (20±2) ps effective time constant for charge separation. The primary
processes in PS II appear too complicated for such a simplistic interpretation of the data. Notice
that the different methods and measurement means were used, however, at the PS reaction center
the main paths of excitation energy to be decayed are the non-radiative and radiative decay com-
peting with the charge separation of excitation energy conversation and stabilization. Again at the
moment, the excited energy transfer process for 1P680* is neglected for some parts of effects to be
offset by each other, so we pay attention to the charge separation process in PS II RC competing
with the deactivation of excited energy by the non-radiative and radiative decay. To figure out the
physical intention between the fluorescence lifetime and the effective time constant of the charge
separation, the η is defined as efficiency converted from the excitation energy to electrochemical
energy in PS II RC as follows:
η = 1/(τ r / (1/ τ r + 1/ τ f)) ×100%,
where τ r = 20 ps for the (20 ± 2) ps model and 3 ps for the 3 ps model, and τ f means the fast fluo-
rescence lifetimes. The values of η for the different chloroplasts are listed in table 3.
In the pseudo ginseng chloroplast the excitation energy conversation efficiency η in PS II RC
is 60% calculated on the basis of the 20 ps charge separation model, which is in good agreement
with the distribution of excitation energy and the quantum balance between two photo systems
and the growth nature of the pseudo ginseng plant, exposing that about a half of excited energy in
the PS II of the pseudo ginseng chloroplast might be deactivated by the non-radiative process and
radiative process during the charge separation. Two important factors, the distribution of absorbed
energy associated with light harvesting Chl a/b proteins and excitation energy converted efficiency
from PS II to PS I, affect the distribution of excitation energy and the quantum balance between
two photo systems. One of two extreme cases is that the balance between two photo systems for
the pseudo ginseng chloroplast is dominated by the distribution of absorbed energy associated
with light harvesting Chl a/b proteins, and the effect of excitation conversion on balance is ne-
glected or cannot be clarified. Another extreme case is that absorbed energy associated with light
harvesting Chl a/b proteins accumulates in PS II and the balance between two photo systems for
the pseudo ginseng chloroplast is just controlled by excitation energy conversion from PS II to PS
I, in which the efficiency is estimated to be about 50% due to the reduced electrochemical energy
connecting with PS I. The values of balance (0.30—0.37) for the pseudo ginseng chloroplast
should be affected together by the two factors. Calculated on the 3 ps charge separation model, for
the pseudo ginseng chloroplast η is 91%, which might not account for the balance of excitation
energy distribution in the pseudo ginseng chloroplast and not accord with the nature of the plant

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ELECTRON & ENERGY TRANSFER IN CHLOROPLASTS
377
according to the view of energetics, although there is a plentiful Chl b of PS II in the pseudo gin-
seng chloroplast. In the water hyacinth chloroplast, the absorbed energy associated with light har-
vesting Chl a/b proteins in PS I is highlighted by accumulating Chl a in PS I and the excitation
energy conversation efficiencies both are 98% in the PS II of the water hyacinth and spinach
chloroplasts calculated on the 3 ps charge separation model. This result could hardly interpret that
the balance of excitation energy distribution between two photo systems in the water hyacinth
chloroplast is different from that in the spinach chloroplast. Throughout the excitation energy
conversion efficiency and the application of the charge separation time constant model, our ex-
perimental results prove reasonable to the 20 ps effective charge separation time constant estab-
lished or to a step about an effective 20 ps time in multi-steps charge separation processes in PS II.
Because the excitation energy conversion efficiency in the water hyacinth chloroplast is smaller,
our experimental results demonstrate that the growth rate for the water hyacinth plan is smaller
than that for the spinach plant. But, due to a plentiful amount of carotenoid molecules in the water
hyacinth chloroplast, the capacity of absorbing and trapping light is improved. The biggest value
of η is 91% in PS II RC of the spinach chloroplast, which is clearly distinct from what is always
assumed to have 100% excitation energy conversion efficiency in PS II RC. As the same argument
as mentioned above, we may obtain an estimate that plants can perform highly efficient transfer of
photo-excitation energy from the light-harvesting pigment system to the reaction center (near
100%) due to two factors, the slow fluorescence lifetime and fast excited energy transfer.
Table 3 Excitation energy conversation efficiency [η = 1/(τr (1/τr + 1/τf)) ×100%] in PS II RC for the pseudo
ginseng, water hyacinth and spinach chloroplasts
Pseudo ginseng
Water hyacinth
Spinach
τr/ ps
0—4.0 ns 0—4.5 ns 0—5.0 ns 0—4.0 ns 0—4.5 ns 0—5.0 ns 0—4.0 ns 0—4.5 ns 0—5.0 ns
20
3
59.2
90.6
60.0
90.9
60.8
91.2
87.1
97.8
87.3
97.9
87.7
97.9
90.7
98.5
90.5
98.4
91.1
98.6
4
Conclusions
Having studied the absorption spectra, low temperature steady-state fluorescence spectro-
scopy and the fluorescence lifetime for the isolated chloroplasts, we can get several important
suggestions: (i) These plants all may efficiently absorb light photon. (ii) The fluorescence decays
in PS II measured at the natural QA states for the chloroplasts have been analyzed by a
three-exponential kinetic model and a definite assignment of fluorescence components. The slow
lifetime fluorescence component is assigned to a collection of associated light harvesting Chl a/b
proteins, the fast lifetime component is attributed to the reaction center of PS II and the middle
+
lifetime component may be in agreement with the delay fluorescence of recombination of P680
and Pheo−. Such an assignment of fluorescence component in this paper has taken on comparabil-
ity with the actual models but cherishes more distinct physical significance. (iii) The excitation
energy conversation efficiency in PS II RC is defined and measured. The value of η calculated on

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the same charge separation model is varied for the different chloroplasts. On the 20 ps model, the
values of η are 60%, 87% and 91% for the pseudo ginseng, water hyacinth and spinach chloro-
plasts, respectively. This interesting result is not in conformity to what is assumed that the effi-
ciency is 100% in PS II RC. But, our results show that those plants can perform highly efficient
transfer of photo-excitation energy from the light-harvesting pigment system to the reaction center.
From the application of the effective charge separation time constants, our results may testify the
(20±2) ps model with more rationality, and yet are not likely to obviate the 3 ps effective charge
separation time constant. (iv) The nature of green plant growth may be expressed in the primary
processes of photosynthesis. In the pseudo ginseng chloroplast isolated using the same preparation
as the spinach chloroplast, there is not only a red shift about 10 nm for the fluorescence emission
coming from PS I, but also a discordant balance of excitation energy distribution between two
photo systems. Furthermore, the excitation energy conversation efficiency in PS II RC calculated
on the 20 ps charge separation time constant for the pseudo ginseng is 60%, which falls in with the
nature of the plant. Our experimental results may imply a relationship between a green plant
growth and its fluorescence characteristics and fluorescence lifetime. To maintain highly efficient
photosynthesis, it is necessary for green plant to contain a proportional photo quantum balance
between two photo systems, efficient transfer of photo-excitation energy from the light-harvesting
pigment system to the reaction centers and efficient primary charge separation with high excita-
tion energy conversation efficiency. The slower the plant grows, the less the excitation energy con-
versation efficiency is satisfied.
Acknowledgements The authors thank Prof. Xia Zongju in Peking University and Prof. Peng Hangcheng in the Institute
of Biophysics of Chinese Academy of Sciences for their help in carrying out the single photon counting experiment, and Dr. Lin
Su in Photosynthesis Research Center of Arizona State University for presenting the fitting program (ASUFIT) initially edited by
Joel Hindorff and Neal Woodbury. This work was supported by the State Key Basis Research and Development Plan (Grant No.
G1998010100), National Natural Science Foundation of China (Grant Nos. 29733100, 39890390) and Postdoctoral Fund.
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