SCIENCE IN CHINA (Series C)

Vol. 46 No. 5

October 2003

The composition and spectral properties of three different forms of light-harvesting complex II LENG Jing (य

ࡁ), LI Liangbi (ह॥ឫ) & KUANG Tingyun (ࣜඳၩ)

Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China Correspondence should be addressed to Li Liangbi (email: [email protected]) Received March 10, 2003

Abstract Different aggregates of LHC II play a very important role in regulating the light absorption and excitation energy transfer of plant. Trimeric LHC II was purified from spinach thylakoid membrane. In order to obtain the dimeric and monomeric LHC II, the trimer was treated with the mixture of 2% OGP and 10 Pg/mL PLA2, then loaded onto the sucrose density gradient in the presence of 0.06% triton X-100. The LHC II trimer, dimer and monomer isolated by sucrose density gradient all contained three polypeptides with molecular weight of 29, 28 and 26 kd respectively. The pigment composition showed much difference in the content of Chl b and xanthophyll among three forms of LHC II. To study the light capture and excitation energy transfer in different forms of LHC II, the absorption and fluorescence spectra were analyzed. The results clearly showed that the efficiency of energy absorption and transfer was different in the three kinds of LHC II, the highest for trimeric LHC II, intermediate for dimeric LHC II, and the lowest for monomeric LHC II. It was suggested that there might be a physiological homeostasis of different aggregates of LHC II in plants, which is significant for the plant self-regulating upon exposure to variable light environment. Keywords: LHC II, aggregate, pigment, energy absorption and transfer. DOI: 10.1360/02yc0195

Light-harvesting pigment-protein complexes arrayed in the thylakoid membrane serve as antenna to capture light energy and deliver it to photosynthetic reaction centers. The antenna complex of photosystem II (LHC II) is the most abundant pigment-protein complex in green plants. LHC II contains a set of polypeptides encoded by nuclear genes belonging to Lhcb family, of which, LHCB1, LHCB2 and LHCB3, encoded by Lhcb1—3, assemble to form heterotrimer on thylakoid membrane. The LHC II trimer not only absorbs and transfers energy, but also plays a role in the formation of grana stacking and regulating the energy distribution between two photosystems[1—3]. It was reported that the alterable conformation of LHC II could be induced by various light environment, thus leading to the change of the thylakoid membrane microstructure[4]. The aggregation of LHC II trimer can also lead to fluorescence quenching and consequent photoprotection[5,6]. The spectral analysis of pigment-protein complex in relation to their structure is of most importance in understanding the mechanism of the energy conversion in photosynthesis, so the spectroscopic properties of LHC II have been paid much attention recently [7,8].

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It was known that the light-harvesting complex of PSI was a dimer[9]. TrØmoliŁres suggested that aproprotein of LHCB was transported to the thylakoid membrane, then bound by pigments to form the monomeric pigment-protein complex. At last, LHCB1 and LHCB2, together with the minor LHCB3 formed the major trimeric LHC II complex in the presence of membrane lipids[10]. During the formation process of LHC II trimer, there may exist LHC II dimer and monomer. As a matter of factor, researchers have observed the existence of LHC II monomer and aggregates, which also participate in the light harvest and photoprotection under excess light[11,12]. So far, to investigate the composition and function of different forms of LHCII is significant in understanding the mechanism of regulating energy absorption and transfer in photosynthesis. In this work, LHC II trimer was isolated from spinach thylakoid membrane. Then LHC II dimer and monomer were obtained by sucrose gradient centrifugation after the treatment of LHC II trimer with a mixture of OGP and PLA2. Comparison of spectroscopic properties was made among the LHC II trimer, dimer and monomer to understand the differences of them in both of the pigments binding and energy transfer. 1

Materials and methods

1.1

Materials Spinach (Spinacia oleracea L.) used for LHC II isolation was purchased from the market in Beijing. N-Octyl -D-Glucopyranoside (OGP), N-Dodecyl- -D-Maltoside (DM) and Phospholipase A2 (PLA2) were purchased from Sigma Chemical Company. 1.2 Methods 1.2.1 Preparation of trimeric, dimeric and monomeric LHC II. LHC II trimer was isolated [13] and purified from spinach thylakoid membranes by the method of Xu with slight modification. PSII particle was treated with 0.8% Triton X-100 for 15 min, then centrifuged at 40000 g for 10 min. The pellet was solubilized with 35 mmol/L nonionic detergent n-Octyl -D-Glucopyranoside (OGP), followed by centrifugation at 40000 g for 40 min. The pellet was resuspended in buffer containing 20 mmol/L Mes, 15 mmol/L NaCl and 5 mmol/L MgCl2, pH 6.0, then stored at −80℃ for further use. To obtain the dimer and monomer, LHC II trimer was treated with a mixture of 2% OGP and 10 μg/mL phospholipase A2 (PLA2, from bee venom) in 22 mmol/L trishydroxy-methylaminemethane (pH 8.0) at room temperature overnight. The final concentration of chlorophyll in the mixture was 1.4 mg/mL. The sample was then loaded onto a freshly prepared sucrose gradient and centrifuged at 160000 g for 24 h at 4℃. After centrifugation, the green bands in the centrifuge tubes were aspirated carefully and dialysed overnight against buffer containing 20 mmol/L Hepes (pH 7.5), 0.06% (w/v) DM, and 70% (v/v) glycerol[14]. 1.2.2 Preparation of sucrose gradients. To prepare sucrose gradients, centrifuge tubes were filled with 10 mL sucrose gradient mix solution (25 mmol/L Mes, 0.6 mol/L sucrose, 20 mmol/L

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NaCl and 0.06% Triton X-100, pH 6.0) and frozen at 20ć for 8 h, then slow thawing at 4ć resulted in the formation of a sucrose density gradient[15]. 1.2.3 Polyacrylamide gel electrophoresis. The purity and stability of the isolated LHCII preparations were checked by denaturing and native polyacrylamide gel electrophoresis. SDS-PAGE was performed following the method of Laemmli[16], 4% acrylamide in stacking gel and 15% in separation gel containing 6 mol/L urea. The gels were stained with Coomassie brilliant blue R-250. Native-PAGE was performed as described by Schägger[17]. Sample was solubilized in the buffer containing 750 mmol/L aminocaproic acid, 50 mmol/L BisTris/HCl, pH 7.0, 60 mmol/L OGP and 0.1% taurodeoxycholate. The concentrations of acrylamide were 4% in stacking gel and 8% in separation gel. 0.05% Triton X-100 and 0.05% Na-taurodeoxycholate were added in the cathode buffer. 1.2.4 Pigments analysis. To determine the pigment compositions in different LHC II complexes, the LHCII samples were extracted in ice-cold 100% acetone and the pigment extracts were filtered through a 0.45 Pm membrane filter. The chlorophyll concentration and the ratio of Chl a/b were determined according to Arnon[18]. Pigments were separated and quantified by HPLC mainly according to Thayer and Björkman[19]. The detection wavelength was 440 nm. The concentration of protein was determined by UV absorption at 260 nm and 280 nm as the method described by Li[20]. 1.2.5 Spectra measurements. For the measurement of steady-state absorption and 77 K fluorescence spectra, different forms of LHC II were solubilized in 0.4 mol/L sucrose, 0.05% DM, 20 mmol/L Mes, pH 7.0. Absorption spectrum was recorded with an UVKON-943 dual beam spectrophotometer. Fluorescence spectrum was recorded by a Hitachi F4500 fluorescence spectrophotometer. The excitation slim and emission slim were 10 nm and 5 nm respectively. The excitation wavelength was 436 nm for Chl a and 480 nm for Chl b. The concentration of sample was adjusted to 10 Pg Chl/mL for measurement. 2

Results

2.1

Isolation of LHC II in different forms The starting material for all complexes described in this paper was trimeric LHC II isolated from spinach thylakoid membrane. Sucrose gradient centrifugation is a very moderate method to purify samples, so we use it to isolate different forms of LHC II. Since the native LHC II has the tendency to aggregate in solvent, detergent of moderate concentration is needed. When 0.06% DM or 0.08% OGP was added into the sucrose gradient, the trimer aggregated at the bottom of the centrifuge tube, then 0.06% Trinton X-100 was chosen and the sample was separated into three bands in the gradient as shown in fig. 1(a). The result of native-PAGE (fig. 1(b)) showed that, by dealing the trimer with mixture of 2% OGP and 10 Pg/mL PLA2, there appeared an obvious green band of dimer whose position was in the middle of the trimer and monomer bands on the gel. This indicated that three forms of LHC II were isolated successfully.

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Fig. 1.

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Different forms of LHC II isolated by sucrose density gradient (a) and native-PAGE (b). Lane 1, LHC II treated with

mixture of 2% OGP and 10 Pg/mL PLA2; lane 2, native LHC II.

2.2

The polypeptide composition The polypeptide composition of these three forms of LHC II was analyzed by SDS-PAGE, and the result is shown in fig. 2. They all consist of three polypeptides (encoded by Lhcb1—3) with molecular weights of 29, 28 and 26 kD respectively. LHCII trimer is a heterotrimer that assembles randomly in vivo[10]. When the trimer was treated with detergent and enzyme in vitro, one of the three subunits dissociated to form the monomer, while the other two existed in form of dimer. Some of the trimer did not react with the enzyme and detergent, so they maintained the former conformation. Therefore, the results presented in fig. 2 showed that the dimer and mono-

Fig. 2. The SDS-PAGE of different forms of LHC II. Lane 1, Marker; lane 2, trimer; lane 3, dimer; lane 4, monomer.

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mer we isolated were still the mixtures of three polypeptides in their composition. 2.3

The pigment composition The three-dimensional structure of LHC II at 3.4 Å determined by Kühlbrandt[21] suggested that each monomer bound at least 12 Chls and 2 Carotenoids (Cars). The data shown in table 1 are from the pigment analysis of different forms of LHC II. Chl a, Chl b, lutein, neoxanthin and violaxanthin were detected, but no E-carotene was found in these LHC II. The results indicated that LHC II trimer was associated with a greater number of chlorophylls and carotenoids than dimer and monomer. The pigment content in this table was calculated based on protein concentration, so it indicated the quantity of pigment binding at per mg protein. The content of Chl a was about 60 Pg/mg protein in trimer, while it was 55 Pg/mg protein and 40 Pg/mg protein in dimer and monomer. The contents of Chl b, lutein and violaxanthin in dimer were only 61%, 37% and 38% of that in trimer, and the data in monomer was 25%, 28% and 39%. The content of Chl b and neoxanthin in monomer was about half of that in dimer. This suggested that the contents of Chls and carotenoids decreased upon LHC II dissociation. Table 1 Chl a

The pigment composition of different forms of LHC II Chl b

Lutein

Violaxanthin

Neoxanthin

(Pg/mg protein) Trimer

60.06 ± 8.11

46.45 ± 4.72

16.33 ± 2.63

12.08 ± 2.01

3.05 ± 0.58

Dimer Monomer

55.45 ± 3.19 40.33 ± 5.02

28.39 ± 2.64 12.28 ± 2.22

6.00 ± 1.58 4.61 ± 1.29

4.62 ± 1.14 4.69 ± 0.95

3.42 ± 0.43 1.76 ± 0.37

2.4

Fluorescence spectra Figs. 3 and 4 show the fluorescence spectra of the three kinds of LHC II at 77 K. The fluorescence emission band upon excitation at 480 nm (Chl b) is higher than that upon excitation at 436 nm (Chl a) in trimer because of the binding of considerable Chls b, while it is lower than the band excited at 436 nm in dimer and monomer (fig. 3). It indicates that due to the decrease of Chl b in dimer and monomer, the energy transfer efficiency from Chl b to Chl a declines as well. The fluorescence excitation spectrum of LHC II is characterized by two bands at 440 nm and 480 nm as shown in fig. 4. The band at 480 nm is relatively high compared with the band at 440 nm in trimer. Upon dissociation, the band at 440 nm decreases and splits into two bands with wavelength of 474 nm and 483 nm in dimer, 473 nm and 484 nm in monomer. This might be intimately related to the changes in energy transfer from Cars to Chl b and in the interactions between the pigments and proteins. 2.5

Absorption spectra The absorption spectra of different forms of LHC II at room temperature are given in fig. 5. There are two characteristic bands at 437 nm (Chl a) and 475 nm (Chl b) in soret region, as well as other two bands at 655 nm (Chl b) and 675 nm (Chl a) in red region[12]. Compared with trimer, the

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Fig. 3. 77 K fluorescence emission spectra of different forms of LHC II. (a) LHC II trimer; (b) LHC II dimer; (c) LHC II monomer.

amplitude of the bands at 475 nm and 675 nm decrease in dimer and monomer, and the band at 650 nm decreases only in monomer. The results are consistent with the declining contents of Chl b and Chl a in dimer and monomer. In order to further learn the absorption difference among trimer, dimer and monomer, the forth derivative of absorption spectrum (fig. 6) is detected. For trimer, there are four bands at 460 nm and 470 nm (Chl b), 437 nm (Chl a) and carotenoid transition at 480 nm in Soret region[22]. Compared with trimer, there is a clear disappearance of the band at 470 nm. In the red region, the changes in amplitudes of three bands at 650 nm (Chl b), 669 nm and 680 nm (Chl a)[22] are observed. Due to the sharp decrease at the band of 680 nm, the shoulder around 669 nm becomes more obvious in dimer and monomer. In addition, the bands at 650 nm and 680 nm decrease gradually in trimer, dimer and monomer. The forth derivative of absorption spectrum also indi-

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Fig. 4. 77 K fluorescence excitation spectra of different forms of LHC II. (a) LHC II trimer; (b) LHC II dimer; (c) LHC II monomer.

Fig. 5. The absorption spectra (R.T.) of different forms of LHC II.

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cates that the differences in red region of Chl a absorption are obvious, while in Soret region, the differences of Chl b absorption are conspicuous. It also can be concluded that the absorption spectra of dimer and monomer are similar, but they are much different from that of trimer. 3

Discussion

3.1

Compositions of different forms of LHC II In this paper, we have described the biochemical and spectroscopic characterization of the trimeric, dimeric and monomeric LHC II, purified by sucrose density gradient centrifugation after treatment of the native LHC II trimer with mixture of OGP and PLA2. SDS-PAGE result revealed that three different forms of LHC II contained the same three polypeptides (fig. 2). Previous work performed on Arabidopsis and carnation directly demonstrated the existence in vivo of Lhcb(1)3 homotrimers as well as Lhcb(1)2Lhcb2, Lhcb(1)2Lhcb3 and Lhcb1Lhcb2Lhcb3 heterotrimers[23] and it is maybe the same in spinach thylakoid membranes. So the LHC II obtained in this study should be a mixture of different polypeptide composition. In contrast, pigment analyses indicate that LHC II trimer is associated with a greater number of chlorophyll b, lutein and violaxanthin molecules than the dimer and monomer. The reason for difference in the pigment composition is probably that LHC II exists predominantly as a trimer in vivo and that the dimer and monomer are the products of the trimer dissociation induced by detergent and enzyme. According to the pigment locations in LHC II described by van Amerongen[24], two lutein molecules bind the inner sites of the apoprotein, and the outer sites are bound by Chl a and Chl b in turn. Furthermore, there are also some carotenoids in xanthophyll cycle peripherally bound to the complexes. Thus the binding of xanthophyll and Chl b is affected mostly upon trimer dissociation. Our results are in line with this viewpoint. The reason why the pigment contents vary in different forms of LHC II might be ascribed to two aspects. One of them is that during the formation of LHC II trimer in vivo, the monomer associated with less pigment binds several more pigments and then assembles to form dimeric and trimeric aggregates. These pigments play not only functional but also structural roles in trimer and they are prone to dissociate from the protein upon disaggregation. The other may be due to the effects of enzyme and detergent, which destroy the binding of pigment and result in the decrease of pigment content in dimer and monomer. It still needs more evidence to confirm them. 3.2

Energy absorption and transfer in different forms of LHC II The changes in pigments binding and protein conformation lead to the pronounced alternations of the spectroscopic properties. Of the three types of LHC II studied, the combined measurements of absorption and fluorescence spectra provide a sensitive tool for monitoring of the dynamic behavior of LHC II pigments in relation to the transfer and regulation of the excitation energy. It may help us understand why trimeric LHC II is selected for the functional structure for energy absorption and transfer in vivo. In parallel with the decrease of Chl b and xanthophyll in pigment analysis, energy transfer

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from Cars to Chl b and from Chl b to Chl a was affected. The disappearance of the Soret band transition at 470 nm (Chl b) that was most likely caused by the loss of Chl b indicated the decrease in Chl b absorption as well as in the energy transfer from Chl b to Chl a in dimer and monomer (fig. 6). The fluorescence emission and excitation spectra showed obvious decrease of Chl b region in dimer, especially in monomer. The emission peak of 680 nm upon excitation at 480 nm (Chl b) declined in dimer and monomer, which resulted from the decrease in energy transfer from Chl b to Chl a (fig. 3). The excitation peak at 480 nm decreased and was split into two peaks in dimer and monomer, which indicated that the energy transfer from xanthophyll to Chl b was destroyed (fig. 4). Kleima et al.[14] reported that the energy transfer between monomers was mostly carried by Chl a. Although the Chl a content decreased not such much in dimer and monomer, the amplitudes of Chl a absorption bands in red region declined, which consequently affected the energy transfer from Chl a to Chl a between monomers. So it can be concluded that the efficiencies of three excitation energy transfer ways, including Cars-Chl b, Chl b- Chl a as well as Chl a-Chl a decline in dimer and monomer compared with trimer.

Fig. 6. The forth derivative of absorption spectra of different forms of LHC II.

The results described in this paper have revealed that LHC II trimer, dimer and monomer are different in the pigment composition, structure conformation as well as the energy absorption and transfer efficiency. The trimer is the most efficient unit to capture and transfer the light energy in three types of LHC II. It might suggest that plants can modulate the absorption of light energy, and control the excitation energy transfer from LHC II to reaction center (RC) via the physiological homeostasis of different LHC II aggregates in vivo. As a result, the excitation energy transferred to RC is just enough for the light reaction, but not so much to induce the photodamage of RC. This is an interesting question worthy of further study.

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