LI Dong-Hai 1, RUAN Xiang 1, XU Qiang 1, GONG Yan-Dao 1, ZHANG Xiu-Fang 1, ZHAO Nan-Ming 1* WANG Ke-Bin 2, KUANG Ting-Yun 2

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1 Acta Botanica Sinica 2004, 46 (4): A Circular Dichroism Spectroscopic Study Revealing the Cause of the Changes of Chlorophyll a Fluorescence Induction of Photosystem During Heat Treatment LI Dong-Hai 1, RUAN Xiang 1, XU Qiang 1, GONG Yan-Dao 1, ZHANG Xiu-Fang 1, ZHAO Nan-Ming 1* WANG Ke-Bin 2, KUANG Ting-Yun 2 (1. State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing , China; 2. Photosynthesis Research Center, Institute of Botany, The Chinese Academy of Sciences, Beijing , China) Abstract: Chlorophyll a fluorescence and circular dichroism (CD) spectra of photosystem (PS ) 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 PS membranes and LHC (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 LHC in PS is related to the anomalous CD signal, and can be an important factor influencing Fo' in the heat treatment of PS membrane. Key words: chlorophyll; circular dichroism (CD); photosystem (PS ); light-harvesting chlorophyll a/b binding complex (LHC ) 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 interactions, e.g., due to excitonic interactions between molecules on neighboring particles, and a distortion by differential scattering. However, the large aggregation-induced 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 of the macro-helix, is controlled by the volume of the aggregate (Keller et al., 1986). Barzda et al. (1994) have investigated the CD of pigment-protein complexes in different aggregation states both in thylakoid membranes and in isolated LHCII, and provided direct experimental evidence that the magnitude of the major CD bands increases with the s ize of the aggregates. In their experiments, they treated the materials with MgCl 2 and Triton X-100 in order to cause the anomalous 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 information on the various aspects of photosynthesis, and a good non-destructive technique for evaluating the heat-induced effects on photosystem (PS ) 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 both Fv and Fv/Fm have been reported (Yamane et al., 1997; Yamane et al., 1998; Pospíšl et al., 1998). Mechanisms about changes of the chlorophyll a fluorescence 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 lightharvesting chlorophyll a/b binding complex (LHC ) from PS core complex (Schreiber et al., 1978) and inhibition of electron flow from Q A to Q B (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, PS membrane was adopted to study the heat-induced changes of CD and chlorophyll a fluorescence. The results indicated that during the heat treatment of PS membrane anomalous CD signals was strongly related to the aggregation state of LHC which could be a factor influencing the chlorophyll fluorescence parameter Fo'. Received 30 Jun Accepted 20 Oct * Author for correspondence. <dbsznm@mail.tsinghua.edu.cn>.

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4 in s perm heads, condens ed chromatins, and DNA aggregates, respectively. In general thylakoid membranes and in macroaggregates of LHC, nonconservative CD signals with extremely large amplitudes have been observed. In the present experiment, in PS membranes, anomalous CD s ignal with extremely large amplitudes also occurred during the heat treatment. The anomalous increase of CD signal could not 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 PS complexes (Gregory et al., 1980), and attributed to long-range interactions of aggregation (Simidjiev et al., 1997). According to the results shown in Fig. 2, the anomalous CD signals were not induced by the state of aggregation of the PS membrane, but that of the LHC during the heat treatment. Barzda et al. (1994) also observed the Triton X-100-treated anomalous CD of LHC and provided direct experimental evidence of the size dependency of CD in macroaggregates. The results indicated that though the treatments were different in these experiments, the mechanism of the changes of LHC could be accordant. In PS membranes and LHC -enriched complexes, the anomalous CD may be involved in the changes of the aggregation state of LHC. During the heat treatment, the increase of Fo' with the rise of temperature was usually attributed to detachment of LHC from PS core complex (Schreiber et al., 1978) and/or inhibition of electron flow from Q A to Q B (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 might exist to influence Fo' value. Fig. 2a shows that the CD signal rapidly increased at the temperatures from 30 to 40, indicating that the changes of the aggregation state of LCH occurred, in other words, the size of aggregation of LHC in the heat-treated PS could be bigger than that in the native LHC (Barzda et al., 1994; Simidjiev et al., 1997). The increase of the aggregation of LHC could quench its chlorophyll fluorescence (Mullet et al., 1980; Ruban et al., 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 of CD intensity after treatment at temperatures from 40 to 55, indicating the size of aggregation of LHC became smaller, which would increase the fluorescence Fo' value. This is consistent with the results shown in Fig.1. Therefore, our results showed that the s tate of aggregation of LHC can be a factor influencing Fo' in the heat-induced denaturation of PS. During the heat treatment of PS 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. References: Barzda V, Mustárdy L, Garab G Size dependency of circular dichroism in macroaggregates of photosynthetic pigmentprotein complexes. Biochemistry, 33: Berry J, Björkman O Photosynthetic response and adaptation to temperature in higher plants. Ann Rev Plant Physiol, 31: Bukhov N G, Sabat S C, Mohanty P Analysis of chlorophyll a fluorescence changes in weak light heat -t reat ed Amaranthus chloroplast. Photosynth Res, 23: Garab G, Kieleczawa J, Sutherland J C, Bustamante C, Hind G Organization of pigment-protein complexes into macrodomains in the thylakoid membranes of wild type and chlorophyll b-less mutant of barley as revealed by circular dichroism. Photochem Photobiol, 45: Garab G, Wells K S, Finzi L, Bustamante C Helically organized macroaggregates of pigment-protein complexes in chloroplasts: evidence from circular intensit y differential scattering. Biochemistry, 27: Gregory R P F, Demeter S, Faludi-Dániel A Macromolecular organization of chlorophyll a in aggregated chlorophyll a/ b protein complexes shown by circular dichroism at room and cryogenic temperatures. Biochim Biophys Acta, 591: Keller D, Bustamante C Theory of the interaction of light with large inhomogeneous aggregates.. Psi-type circular dichroism. J Chem Phys, 84: Kuwabara T. M urata N Inactivat ion of p hotosynt hetic oxygen evolution and concomit ant release of three polypeptides in the photosystem particles of spinach chloroplasts. Plant Cell Physiol, 23: Livolant F, Maestre M F Circular dichroism microscopy of compact forms of DNA and chromatin in vivo and in vitro: cholesteric liquid-crystalline phases of DNA and single dinoflagellate nuclei. Biochemistry, 27: Lou S Q, Wang K B, Zhao F H, Xu C H, Kuang T Y A comparative study on PS light harvesting chlorophyll a/b protein complexes between spinach and cucumber. Acta Bot Sin, 37: Maestre M F, Bustamante C, Hayes T L, Subirana J A, Tinoco I Jr Differential scattering of circularly polarized light by the helical sperm head from the octopus Eledone cirrhosa. Nature, 298: Mullet J, Arntzen C J Simulation of grana stacking in a

5 LI Dong-Hai et al.: A Circular Dichroism Spectroscopic Study Revealing the Cause of the Changes of Chlorophyll a Fluores cence Induction of Photosystem During Heat Treatment model membrane system:mediat ion by a purified light-harvesting pigment-protein complex from chloroplasts. Biochim Biophys Acta, 589: Pospíšl P, Skotnica J, NaušJ Low and high t emperature dependence of minimum Fo and maximum Fm chlorop hyll fluorescence in vivo. Biochim Biophys Acta, 1363: Reich Z, Ghirlando R, Minsky A Secondary conformational polymorphism of nucleic acids as a possible functional link between cellular parameters and DNA packaging processes. Biochemistry, 30: Ruban A V, Horton P Mechanism of ph-dependent dissipation of absorbed excitation energy by photosynthetic membranes : spectroscopic analysis of isolated light-harvesting complexes. Biochim Biophys Acta, 1102: Schreiber U, Berry J A Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damage of the photosynthetic apparatus. Planta, 136: Schreiber U, Armond P A Heat-induced change of chlorophyll fluorescence in isolated chlorop lasts and relat ed heatdamage at the pigment level. Biochim Biophys Acta, 502: Simidjiev I, Barzda V, Mustárdy L, Garab G Isolation of lamellar aggregates of the light-harvesting chlorophyll a/b protein complex of photosystem with long-range chiral order and structural flexibility. Anal Biochem, 250: Yamane Y, Kashino Y, Koike H, Satoh K Increases in the fluorescence Fo level and reversible inhibition of Photosystem react ion cent er by high-temp erat ure treatments in higher plants. Photosynth Res, 52: Yamane Y, Kashino Y, Koike H, Satoh K Effects of high temperatures on the photosynthetic systems in spinach: oxygen-evolving activities, fluorescence characteristics and the denaturation process. Photosynth Res, 57: (Managing editor: HE Ping)