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1 隐藻藻蓝蛋白与类囊体膜的动态结合模型徐伟, 梁源, 陈敏, 王宁 (, ) 摘要 : 隐藻藻胆蛋白在类囊体腔中的存在状态始终存在争议, 为了深入了解这一问题, 作者以含有藻蓝蛋白 (PC645) 的蓝隐藻 (Chroomonas placoidea) 活体细胞及充分洗涤过的类囊体膜为材料, 测定其室温吸收光谱荧光谱以及 77K 低温荧光光谱, 并根据光谱数据分析了 PC 在类囊体腔中的存在状态结果显示, 蓝隐藻的类囊体膜上始终紧密结合着一定量的 PC, 并且很可能是以 PC 的 β 亚基与膜相接触, 证实藻胆蛋白在类囊体腔中不是完全游离的, 与膜的接触也并非完全无倾向性排布 ; PC 既可将激发能传递给光系统 (PS)II, 但也可传递给 PSI, 相较于叶绿素 (Chl)a 和 Chl c, 在 PSI 长波荧光中 PC 的贡献更明显, 而 Chl c 吸收的光能, 更倾向于传递给 PSII 或者短波长 Chl a; 蓝隐藻细胞中表现出超比例的 PC 荧光发射, 说明除了结合在膜上的 PC 外, 有一部分 PC 是游离的, 其存在对于隐藻捕光功能来说可能是有冗余的 ; 游离的 PC 可与类囊体膜重新接触而恢复能量传递功能根据实验结果以及前人的报道, 提出了隐藻藻胆蛋白与类囊体膜的结合是动态的观点, 并给出了相应的模型关键词 : 蓝隐藻 (Chroomonas placoidea); 藻蓝蛋白 ; 77K 荧光光谱 ; 能量传递 ; 动态结合模型中图分类号 : Q946.1 文献标识码 : A 文章编号 : (2015) doi: /hykx (light-harvesting complex, LHC), LHC (Chl)a/b- Chl a/c-,, [1], Chl a/c 2 -,, : (Cr-PE) (Cr-PC), (APC); (α 1 α 2 ββ), ;, [2-3], : (1) [4] ; (2), [5] ; (3), II(PSII) [6] ; (4), Chl- [7-8],,, Chl a/c 2 - PSI PSII, PC645 (Chroomonas placoidea),, 1 材料与方法 1.1 蓝隐藻的培养及藻体细胞的收集 F/2, 20~25,, 16W, lx 4~5 d, g 10 min, (ph mmol/l, 0.3 mol/l, 0.1 mol/l NaCl MgCl 2 ) : ; : : ( ) : (1990-),,,,, : , sky_x1227@163.com;,, chenmclm@163.com Marine Sciences / Vol. 39, No. 4 /

2 1.2 蓝隐藻类囊体膜的制备 (50 mmol/l, ph8.0 Tricine-NaOH), French Pressure Cell Press( SLM Aminco), MPa g 10 min, g 10 min,, 2~3, PC, 50%, Jeffrey [9] 0.8~1.5 g/l, 藻蓝蛋白 PC645 的制备 g 10 min, PC645 50%, 80% PC645, g 15 min, PBS Sephadex G-100, 50 μl/l PBS PC 室温吸收光谱测定, TU-1900 ( ) 1.5 室温荧光光谱测定, LS55 (Perkin Elmer) 500 nm/min, 10.0 nm K 荧光光谱测定, min, g 10 min 80% / (V/V),, g 15 min PC Laemmli [10] SDS-PAGE, 10%~20%, 7%, 0.5 mol/l NaCl 2 实验结果 2.1 吸收光谱 ( 1), 436(438)nm 677(679) nm Chl a; 495 nm ; nm nm PC( nm), Cr-PC nm Chl c, (630 nm ) PC645, PC Chl a, PC, Chl c PC, Chl a, PC Chl c,, Chl a [11] ( 2)Chl a 679 nm, 580~650 nm PC ( nm ), Chl a, PC, 60%, 60%(V/V),, LS nm/min, 15.0 nm, 10.0 nm; 20.0 nm, 10.0 nm 1.7 SDS-PAGE,, PC,, 0.8 g/l Laemmli [10] Fig.1 1 Absorption spectra of cells and PC645 at room temperature 22 / 2015 / 39 / 4

3 2 Fig.2 Absorption and derivative spectra of thylakoids at room temperature, PC,,, 2.2 藻体室温荧光光谱 3A, 435 nm ( Chl a ) 460 nm( Chl c ), PSII Chl a 686~687 nm, 640 nm Chl c, Chl c 100% Chl a Chl c Chl a, Chl a/c-,,, Chl- 580 nm( PC ), 686 nm Chl a PC Chl a Chl a 580 nm, 580 nm Chl a 435 nm ( 1.5~1.8 ), Chl a PC Chl a, 580 nm 1 662~667 nm PC, Chl a (F667/F686=1.05~1.2), PC Chl a,,, Chl a, 98%~99% [7], 660~667 nm PC,,, [12] 3B, 700 nm,, nm( nm) Chl a Chl c, 679 nm Chl a, Chl c Chl a,, 582 nm 646 nm( , 631 nm 625 nm ) PC, 700 nm PC 520~550 nm( 529 nm 543 nm), PC [13-14] 2.3 藻体 77K 荧光光谱 PSII, PSI [15], 77K, PSI PSII 77K, ( 3C),, : (1) 435 nm 460 nm, Chl a 694~696 nm,, PSII [16], PC Chroomonas CCMP270 PE Rhodomonas CS24 77K PSII nm, PSII [7, 17],,, PSII, 700 nm, Chl a Chl c PSII Chl a Chl c Chl a, 77K Chl c Chl a (2) 580 nm PC, 695 nm PSII, 720~725 nm, PSI [18-19] Mimuro [16] Marine Sciences / Vol. 39, No. 4 /

4 3 (A, B) 77K(C, D) (A, C), (B, D) Fig. 3 Fluorescence emission (A, C), excitation (B, D) and derivative spectra of cells at room temperature (A, B) and77 K (C, D) A C. 435nm( ), 460nm(------), 580nm( ), 580nm ( ); B. 700nm( ), ( ); D. 720nm( ), ( ) A, C. Excitation wavelength 435nm( ), 460nm(------), 580nm( ), derivative spectra at 580nm( ); B. Em = 700nm( ), derivative spectra( ); D. Em= 720nm( ), derivative spectra( ) PSI 77K 715~730 nm PC PSII PSI Chl a 700 nm PC, PC Chl a (3) 580 nm 666 nm, PC, PSI PSII Chl a (F666/F , F666/F725=6.0), Chl a 4~5, PC [7, 17], [20-21] PC, PC, 666 nm Chl a, PC,, PC, 720 nm 77K ( 3D), 440~470 nm Chl a Chl c, 687nm Chl a 580~650 nm, PC645 ( 1) 77K PC, nm PC, PC645 3,, 4 (A, B) 77K(C, D) (A, C), (A, C) Fig.4 Fluorescence emission (A, C), excitation (B, D) and derivative spectra of thylakoid membrane at room temperature(a, B) and 77 K (C, D) A C. 435nm( ), 460nm(------), 580nm( ); B. 700nm( ), ( ); D. 720nm( ), ( ) A, C. Ex= 435nm( ), 460nm(------), 580nm( ); B. Em = 700nm( ), derivative spectra( ); D. Em= 720nm( ), derivative spectra( ) 24 / 2015 / 39 / 4

5 PC645 : DBV( ) 580~585 nm, MBV( ) 610~622nm, PCB( ) 630~640 nm, PCB(β82) 645~650 nm, β 82 Cys PCB [22-23] 4 DBV(581 nm) MBV(611 nm) PCB(632 nm) PCBβ82(649 nm) 2.4 类囊体膜室温荧光光谱 nm, 688~689 nm PSII Chl a ( 4A), Chl a Chl c, Chl a 580 nm, 685~686 nm Chl a,, PC, 435 nm ( 0.2~0.3), Chl c ( 0.7~0.8), 659~660 nm ( 5~6 nm) 1 PC, Chl a F660/F685= 1.4~1.6 Chl- PC 700 nm ( 4B), PC, Chl a, 435 nm, 673 nm ; 460 nm Chl c, Chl c ; 583, 632, 642 nm PC, PC, PC Chl a 2.5 类囊体膜 77K 荧光光谱 4C 77K,, 435 nm 460 nm, Chl a Chl c, PSII Chl a 692 nm ( 3~4 nm); 700 ~730 nm, PSI P700, PSI, Chl a Chl c PSI, PSI Chl a/c 2 LHCI [24-25],, LHCI Chl a Chl c,, 580 nm, 662 nm, PSI nm Chl a ( 7 ~8 nm), PC Chl a, Chl a Chl c 4D 77K, 720 nm, Chl a(431~448 nm 682 nm) PC(580~646 nm), Chl c(470 nm) (494 nm) 77K PC, nm, nm PC,, PC, PC, Chl - PC 2.6 类囊体膜多肽 SDS-PAGE 5 : 20~22 ku ( ) 28~35 ku( ) 45 ku (PSII ) 66 ku (PSI )4 [25] ( 5b) ( 5c), 20 ku, PC β ( 5d) /,,, PAGE, PC, ( PC), PC645 β, α ( ), α, PC645 β, PC645 (662 nm ) β PCBβ82, PC645 PCBβ82, Chl a [22-23],, PC645 β, Marine Sciences / Vol. 39, No. 4 /

6 5 PC SDS-PAGE Fig.5 The SDS-PAGE profiles of thylakoid membrane peptides and PC subunits a. (ku); b. ; c. ; d.pc a. standards(ku); b.thylakoid stained by Coomassie brilliant blue under natural light;c.thylakoid stained by ZnSO4 under ultraviolet ray; d.pc stained by Coomassie brilliant blue under natural light β,, 3 讨论与结论, [22-24, 26-28],, Chl-,,, Wit [7],, α 1 α 2 ββ, ;, Chl- ; Föster, Chl a,, Doust [8],, Lichtlé [25] PE- -PSII ; PC-Chl a/c 2 -, PC Chl a, Chl c 2 [29], PSII Chl a/c-,,, Cryptomonas ovata, PSII, PE Chl a/c 2 PSII, PSII, PSII [30], [3], PSI PSII,, [7-8], :, [30-31], ;, PSI PSII,,,, : (1) PC,, PC ; (2)SDS- PAGE, PC645 β, PC645 β, PC645 β α [22-23, 26], PC ; (3)PC PSII, PSI, PC, 26 / 2015 / 39 / 4

7 [17, 22] ; (4) Chl a Chl c, PC PSI, Chl c, PSII Chl a; (5) PC, PC, PC,, :, ;,, (Morisset [32] PC645 ),,,, ; PSI PSII, PSII LHCII Chl a/c, Chl a Chl c, Chl c ; PSI, Chl a ;, LHCII,, : [1] Watanabe M, Ikeuchi M. Phycobilisome: architecture of a light-harvesting supercomplex[j]. Photosynth Res, 2013, 116(2-3): [2] Glazer A N, Wedemayer G J. Cryptomonad biliproteinsan evolutionary perspective[j]. Photosynth Res, 1995, 46(1-2): [3] Gantt E, Edwards M R, Provasoli L. Chl oroplast structure of the Cryptophyceae. Evidence for phycobiliproteins within intrathylakoidal spaces[j]. J Cell Biol, 1971, 48(2): [4] Ludwig M, Gibbs S P. Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens[j]. J Cell Biol, 1989, 108(3): [5] Lichtlé C, Mckay R M L, Gibbs S P. Immunogold localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids[j]. Biology of the Cell, 1992, 74(0): [6] Ingram K, Hiller R G. Isolation and characterization of a major Chl orophyll a/c 2 light-harvesting protein from a Chroomonas species (Cryptophyceae)[J]. Biochim Biophys Acta - Bioenergetics, 1983, 722(2): [7] van der Weij-De Wit C D, Doust A B, van Stokkum I H, et al. How energy funnels from the phycoerythrin antenna complex to photosystem I and photosystem II in cryptophyte Rhodomonas CS24 cells[j]. J Phys Chem B, 2006, 110(49): [8] Doust A B, Wilk K E, Curmi P M G, et al. The photophysics of cryptophyte light-harvesting[j]. Journal of Photochemistry and Photobiology A: Chemistry, 2006, 184(1-2): [9] Jeffrey S, Humphrey G. New spectrophotometric equations for determining Chl orophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton[j]. Biochem Physiol Pflanzen, 1975, 167: [10] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227(5259): [11] Green B R, Parson W W. Advances in photosynthesis and respiration Volume13: Light-harvesting antennas in photosynthesis[m]. The Netherlands: Kluwer Academic Publisher, 2003: [12],. [M]. :, 2000: [13] Maccoll R, Williams E C, Eisele L E, et al. Chromophore topography and exciton splitting in phycocyanin 645[J]. Biochemistry, 1994, 33(21): [14] Maccoll R, Eisele L E, Marrone J. Fluorescence polarization studies on four biliproteins and a bilin model for phycoerythrin 545[J]. Biochim Biophys Acta, 1999, 1412(3): [15] van der Weij-De Wit C D, Ihalainen J A, van Grondelle R, et al. Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy[j]. Marine Sciences / Vol. 39, No. 4 /

8 Photosynth Res, 2007, 93(1-3): [16] Mimuro M, Tamai N, Murakami A. Multiple pathways of excitation energy flow in the photosynthetic pigment system of a cryptophyte, Cryptomonas sp. (CR-1)[J]. Phycological Research, 1998, 46(3): [17] van der Weij-De W C, Doust A B, van Stokkum I H M, et al. Phycocyanin sensitizes both photosystem I and photosystem II in cryptophyte Chroomonas CCMP270 cells[j]. Biophys J, 2008, 94(6): [18] Wientjes E, van Stokkum I H, van Amerongen H, et al. Excitation-energy transfer dynamics of higher plant photosystem I light-harvesting complexes[j]. Biophys J, 2011, 100(5): [19] Kuang T Y, Argyroudi-Akoyunoglou J H, Nakatani H Y, et al. The origin of the long-wavelength fluorescence emission band (77 degrees K) from photosystem I[J]. Arch Biochem Biophys, 1984, 235(2): [20] Mcconnell M D, Koop R, Vasil'Ev S, et al. Regulation of the distribution of Chl orophyll and phycobilinabsorbed excitation energy in cyanobacteria. A structure-based model for the light state transition[j]. Plant Physiol, 2002, 130(3): [21] Joshua S, Mullineaux C W. Phycobilisome diffusion is required for light-state transitions in cyanobacteria[j]. Plant Physiol, 2004, 135(4): [22] Marin A, Doust A B, Scholes G D, et al. Flow of excitation energy in the cryptophyte light-harvesting antenna phycocyanin 645[J]. Biophys J, 2011, 101(4): [23] MacColl R, Williams E C, Eisele L E, et al. Chromophore topography and exciton splitting in phycocyanin 645[J]. Biochemistry, 1994, 33(21): [24] Janssen J, Rhiel E. Evidence of monomeric photosystem I complexes and phosphorylation of Chl orophyll a/c-binding polypeptides in Chroomonas sp. strain LT (Cryptophyceae) [J]. Int Microbiol, 2008, 11(3): [25] Lichtlé C, Duval J C, Lemoine Y. Comparative biochemical, functional and ultrastructural studies of photosystem particles from a Cryptophycea: Cryptomonas rufescens; isolation of an active phycoerythrin particle[j]. Biochim Biophys Acta (BBA) - Bioenergetics, 1987, 894(1): [26] Novoderezhkin V I, Doust A B, Curutchet C, et al. Excitation dynamics in phycoerythrin 545: modeling of steady-state spectra and transient absorption with modified redfield theory[j]. Biophys J, 2010, 99(2): [27] Collini E, Wong C Y, Wilk K E, et al. Coherently wired light-harvesting in photosynthetic marine algae at ambient temperature [J]. Nature, 2010, 463: [28] Huo P F, Coker D F. Theoretical study of coherent excitation energy transfer in cryptophyte phycocyanin 645 at physiological temperature [J]. J Phys Chem Lett, 2011(5): [29] Chen M, Li S H, Sun L. A novel phycocyanin-chl a/c 2 -protein complex isolated from Chl oroplasts of Chroomonas placoidea[j]. Chinese Chemical Letters, 2007, 18: [30] Synder U K, Biggins J. Excitation-energy redistribution in the cryptomonad alga Cryptomonas ovata [J]. Biochim Biophys Acta - Bioenergetics, 1987, 892: [31] Bruce D, Biggins J, Steiner T. Excitation energy transfer in the cryptophytes. Fluorescence excitation spectra and picosecond time-resolved emission spectra of intact algae at 77K [J]. Photochem Photobiol, 1986, 44(4): [32] Morisset W, Wehrmeyer W, Schirmer T, et al. Crystallization and preliminary x-ray diffraction data of the cryptomonad biliprotein phycocyanin-645 from a Chroomonas spec[j]. Archives of Microbiology, 1984, 140(2-3): / 2015 / 39 / 4

9 The dynamic combination model of cryptomonad phycocyanin and thylakoid XU Wei, LIANG Yuan, CHEN Min, WANG Ning (Institute of Life Science, Yantai University, Yantai , China) Received: Sep., 3, 2014 Key words: Chroomonas placoidea; phycocyanin; 77 K fluorescence spectrum; energy transfer; dynamic combination model Abstract: The existential states of cryptomonad phycobiliproteins in thylakoidal lumen have always been controversial. In order to elucidate this question further, the absorption spectra as well as fluorescence spectra (at both room temperature and 77 K) of intact cells and thylakoids of Chroomonas placoidea containing phycocyanin (PC) were determined in this paper. The existential states of PC within thylakoid lumen are discussed according to the data from spectral analysis. The results show that a portion of PC tightly combined to the thylakoid membrane at any time (possibly attached with its β subunit), suggesting that the cryptomonad phycobiliproteins are neither fully isolated across the thylakoid lumen, nor completely have no preference in contacting with thylakoid, and PC functionally transfers excited energy to both photosystem (PS) I and PSII, and contributes more to the long-wavelength of fluorescence from PSI compared to Chlorophyll (Chl ) a and Chl c, while Chl c prefers to transfer excited energy to PSII or short-wavelength of Chl a. The observation of ultrahigh ratio of fluorescence emitted by PC in intact cells indicates the existence of free PC, which may be redundant for light-harvesting. These freely existed PC may associate with thylakoid and revive their light-harvesting function in due time. Considering the results above and those from previous studies, we raise such a point of view that the combination between cryptomonad phycobiliproteins and thylakoid are dynamic, and a corresponding model is described here. ( 本文编辑 : 谭雪静 ) Marine Sciences / Vol. 39, No. 4 /

% GIS / / Fig. 1 Characteristics of flood disaster variation in suburbs of Shang

% GIS / / Fig. 1 Characteristics of flood disaster variation in suburbs of Shang 20 6 2011 12 JOURNAL OF NATURAL DISASTERS Vol. 20 No. 6 Dec. 2011 1004-4574 2011 06-0094 - 05 200062 1949-1990 1949 1977 0. 8 0. 03345 0. 01243 30 100 P426. 616 A Risk analysis of flood disaster in Shanghai