Intrinsic emission properties of trinucleotides at 293 and 77 K

The intrinsic emission properties of two pairs of isomeric trinucleotides, ApCpGp-CpApGp and ApUpGp-UpApGp, in neutral pH solvent between 293 and 77 K were studied. On the basis of the results obtained upon their emission spectroscopy, the fluorescence of these trinucleotides both at 293 and 77K can be regarded as the intramolecular excimer fluorescence which is red-shifted and broadened compared with that of corresponding equimolar mixture of constituent mononucleotides. On the other hand, the phosphorescence of two pairs of isomers observed at 77K is a characteristic of adenine. Thus, it is assumed that in above trinucleotides, the electronic excitation energy transfer takes place via the excimer state to the lowest triplet state of adenine.     

Chlorophyll fluorescence emission spectroscopy of oxygenic organisms at 77 K

Photosynthetic fluorescence emission spectra measurement at the temperature of 77 K (–196°C) is an often-used technique in photosynthesis research. At low temperature, biochemical and physiological processes that modulate fluorescence are mostly abolished, and the fluorescence emission of both PSI and PSII become easily distinguishable. Here we briefly review the history of low-temperature chlorophyll fluorescence methods and the characteristics of the acquired emission spectra in oxygen-producing organisms. We discuss the contribution of different photosynthetic complexes and physiological processes to fluorescence emission at 77 K in cyanobacteria, green algae, heterokont algae, and plants. Furthermore, we describe practical aspects for obtaining and presenting 77 K fluorescence spectra.     

PSII核心复合物能量传递的飞秒时间分辩荧光光谱学研究

运用稳态、瞬态荧光光谱技术对光系统Ⅱ核心复合物的能量传递动力学进行研究。分别用436nm光脉冲激发叶绿素a分子、451nm光激发叶绿素a和β-胡萝卜素分子、473和481nm光激发β-胡萝卜素分子,得到5组反应能量传递、电荷重组等过程的寿命组分：8～40 ps为核心天线中β-胡萝卜素分子通过相邻β-胡萝卜素分子或中间叶绿素a向叶绿素a分子传递能量的时间;85～152 ps为核心天线色素分子激发能传递时间;201～925ps反映部分电荷重组过程;1．031～1．21ns为参与能量传递的色素分子从激发态衰退回到基态的时间;6．17～18、13 ns的长寿命时间组分归因于P680^ Pheo^-的重组过程。将荧光发射谱进行高斯解析,发现在核心复合物中还至少存在Chla683^685、Chla680^682、Chla673,677^679三种特征叶绿素a分子。     

杂色藻类硅藻和金藻的77K荧光特性

实验测定了7种杂色藻包括:4种硅藻三角褐指藻(Phaeodactylum tricornutum)、牟氏角毛藻(Cheatoceros mulleri)、中肋骨条藻(Skeletonema costatum)、尖刺拟菱形藻(Pseudo-nitzschia pungens)和3种金藻湛江等鞭金藻(Isochrysis zhanjiangensis)、绿色巴夫藻(Pavlova viridis)、球形棕囊藻(Phaeocystisglobosa)的77 K荧光光谱,并与其它杂色藻以及高等植物菠菜进行了比较。结果表明,4种硅藻和3种金藻的77 K低温荧光发射光谱相似,只有一个明显的荧光发射主峰位于684~686 nm,类囊体膜的荧光光谱中在700 nm附近有个不明显的肩峰,它们均没有高等植物作为PSⅠ特征的730 nm长波荧光发射峰,这个结果与杂色藻类褐藻的荧光特性一致。说明,杂色藻类PSⅠ缺少730 nm长波荧光峰可能具有其普遍性,预示杂色藻类在PSⅠ结构以及能量传递等方面与高等植物具有很大的差异。     

Energy transfer among the chromophores in phycocyanins measured by picosecond kinetics.

Energy-transfer processes in the algal light-harvesting proteins, the phycocyanins, have been studied by means of picosecond absorption spectroscopy. After excitation at 530 nm, the absorption at several wavelengths in the range 480--669 nm decayed with a short time constant (picosecond) and a long time constant (greater than 1 ns). For C-phycocyanin, energy transfer from the beta to the alpha subunits is interpreted as being a likely candidate for the short time constant; the long time constant probably is the excitation lifetime of the chromophore on the alpha subunits. The time constants for energy transfer in monomers, trimers, and hexamers of C-phycocyanin extracted from a blue-green alga, Phormidium luridum, were measured as approximately 85, approximately 56, and approximately 32 ps, respectively. The corresponding time constant in the cryptomonad phycocyanin 645 from Chroomonas species was found to be less than 5 ps.     

Crystal structure of R-phycocyanin and possible energy transfer pathways in the phycobilisome.

The crystal structure of R-phycocyanin from Polysiphonia urceolata (R-PC-PU) at 2.4 A is reported. The R-PC-PU crystal belongs to space group P4(3)2(1)2 with cell parameters a = 135.1 A, c = 210.0 A, and alpha = beta = gamma = 90 degrees. The structure was determined by molecular replacement. The crystallographic R-factor of the refined model is 0.189 (R(free) = 0.239). Comparison of the microenvironment of chromophore beta 155 in R-PC-PU and in C-PC from Fremyolla diphosiphon (C-PC-FD) reveals that their spectral differences may be caused by their different alpha 28 residues. In the R-PC-PU crystal structure, two (alpha beta)(3) trimers assemble face to face to form a hexamer, and two such hexamers assemble in two novel side-to-side arrangements. Possible models for the energy transfer from phycoerythrin to phycocyanin and from phycocyanin to allophycocyanin are proposed based on several phycobiliprotein crystal structures.     

Gloeobacter violaceus— investigation of an unusual photosynthetic apparatus. Absence of the long wavelength emission of photosystem I in 77 K fluorescence spectra

The deeply purple cyanobacterium Gloeobacter violaceus is subject of this investigation. It does not contain thylakoids, and the photosynthetic apparatus is located in the only membrane of the cell, the plasma membrane. Upon excitation with blue light, the 77 K fluorescence emission spectra of neither intact cells (excited with 427 nm) nor of the isolated plasma membrane (excited with 430 nm), show the expected long wavelength photosystem I emission characteristic for low energy chlorophylls. Maximal fluorescence emission was observed at 688 nm. independent on the excitation wavelength, 427 (430) nm blue light, exciting mainly chlorophyll, or 550 nm green light, exciting mainly phycoerythin. The ratio of P700 to chlorophyll was 175. O₂-evolution was 160 μmol mg_-1 chlorophyll h_-1 in saturating white light; the compensation point was reached at 6 μmol m₂ s_-1 in cultures grown at 25 μmol m₂ s_-1. Dark O₂ uptake was 50 μmol mg_-1 chlorophyll h_-1. During adaptation to increasing white light intensities Gloeobacter reduces the amount of phycocyanin and chlorophyll per cell and strongly increases the concentration of carotenoids relative to chlorophyll. The carotenoid concentration per cell increases with increasing light intensity. Apparently, part of the carotenoids is not located in the plasma membrane.     

The origin of the long-wavelength fluorescence emission band (77 degrees K) from photosystem I

Isolated photosystem I (PSI)-110 particles, prepared using a minimal concentration of Triton X-100 [J. E. Mullet, J. J. Burke, and C. J. Arntzen (1980) Plant Physiol. 65, 814-822] and further subjected to short-term solubilization with sodium dodecyl sulfate (SDS), were resolved into four pigment-containing bands on polyacrylamide gel electrophoresis (PAGE). We have identified these in order of increasing electrophoretic mobility as being (a) CPIa, (b) CPI, (c) the light-harvesting complex of photosystem I (LHC-I), and (d) a free pigment-zone. LHC-I had an absorption maximum in the red at 668-669 nm and a shoulder at 650 nm, which was resolved by its first-derivative spectrum to indicate the presence of chlorophyll b. LHC-I exhibited a 77 degrees K fluorescence emission maximum at 729-730 nm. The 77 degrees K fluorescence emission maxima of CPIa and CPI, excised from the gel, were at 729 and 722 nm, respectively. The LHC-I band, excised from the gel and rerun on dissociating SDS-PAGE, was resolved into two polypeptide doublets of 24-22.5 and 21-20.5 kDa. The CPIa band under similar conditions was resolved into polypeptides of 68, 24, 22.5, 21, 20.5, 19, 15, and 14 kDa; on the contrary, CPI contained only the 68-kDa polypeptide. When intact thylakoids were subjected to "nondenaturing" SDS-PAGE, LHC-I comigrated with an oligomeric form (dimer) of the light-harvesting chlorophyll a/b pigment-protein that preferentially serves photosystem II (LHCP-II). When this combined LHC-I/LHCP-II pigment-protein band was prepared by SDS-PAGE from isolated stroma lamellae, it exhibited a long-wavelength fluorescence band near 730 nm at 77 degrees K. When a similar preparation was obtained from sucrose density gradients containing SDS [J. Argyroudi-Akoyunoglou and H. Thomou (1981) FEBS Lett. 135, 171-181], it was found to be enriched in a 21-kDa polypeptide. The data suggest that the 21-kDa polypeptide of LHC-I is the chlorophyll-containing polypeptide responsible for the long-wavelength fluorescence of LHC-I; other polypeptides in the complex (20.5, 22.5, and 24 kDa) presumably bind chlorophyll and also serve an antennae function.     

Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins

Photon yields of oxygen evolution at saturating CO₂ were determined for 44 species of vascular plants, representing widely diverse taxa, habitats, life forms and growth conditions. The photonyield values on the basis of absorbed light ( ^a) were remarkably constant among plants possessing the same pathway of photosynthetic CO₂ fixation, provided the plants had not been subjected to environmental stress. The mean ^a value ±SE for 37 C₃ species was 0.106±0.001 O₂·photon^-1. The five C₄ species exhibited lower photon yields and greater variation than the C₃ species ( ^a=0.0692±0.004). The ^a values for the two Crassulaceanacid-metabolism species were similar to those of C₃ species. Leaf chlorophyll content had little influence on ^a over the range found in normal, healthy leaves. Chlorophyll fluorescence characteristics at 77 K were determined for the same leaves as used for the photon-yield measurements. Considerable variation in fluorescence emission both at 692 nm and at 734 nm, was found 1) among the different species; 2) between the upper and lower surfaces of the same leaves; and 3) between sun and shade leaves of the same species. By contrast, the ratio of variable to maximum fluorescence emission at 692 nm (F_v/F_M, 692) remained remarkably constant (The mean value for the C₃ species was 0.832±0.004). High-light treatments of shade leaves resulted in a reduction in both ^a and the F_v/F_M, 692 ratio. The extent of the reductions increased with time of exposure to bright light. A linear relationship was obtained when ^a was plotted against F_v/F_M, 692. The results show that determinations of the photon yield of O₂ evolution and the F_v/F_M, 692 ratio can serve as excellent quantitative measures of photoinhibition of overall photosynthetic energy-conversion system and of photochemistry of photosystem II, respectively. This is especially valuable in field work where it is often impossible to obtain appropriate controls.Abbreviations and symbols CAM Crassulacean acid metabolism - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F_o, F_M, F_v instantaneous, maximum, variable fluorescence emission - absorptance - ^a photon yield (absorbed light) - ⁱ photon yield (incident light) C.I.W.-D.P.B. Publication No. 923     

Possible influence of the rate of specimen cooling on the determination of energy distribution in photosynthesis by fluorescence emission at 77 K

The rate of cooling to 77 K appears to be a determining factor in obtaining valid low temperature emission spectra of photosynthetic organisms. Evidence is shown that the usual method of cooling the algae or chloroplasts in suspension leads to artefacts in the spectra and considerable discrepancies in quantitative determinations.     

Connection between the rate of cooling and fluorescence properties at 77 K of isolated chloroplasts

Cooling of chloroplasts to ?196°C can under certain circumstances lead to an erroneous analysis of energy distribution. After minimizing influences of sample geometry and effects of plastid concentration it is shown that externally induced membrane change leads to an increase in the ratio $\text{F}{}_{740}\text{F}{}_{687}$ of the fluorescence emission spectrum. Similar alterations can be observed by variation of the rate of cooling the plastids to 77 K, especially if whole chloroplasts are used. The differences in emission ratios are indicative also of changes in initial energy distribution between the photosystems, given here by the value α_N. This is inferred from experiments with either osmotically induced thylakoid disturbances or those effected through a slow cooling process. The circumstances and the significance of these observations are discussed.     

Quantitative analysis of 77K fluorescence emission spectra in Synechocystis sp. PCC 6714 and Chlamydomonas reinhardtii with variable PS I/PS II stoichiometries

Low-temperature (77 K) fluorescence emission spectra of intact cells of a cyanobacterium, Synechocystis sp. PCC 6714, and a green alga, Chlamydomonas reinhardtii, were quantitatively analyzed to examine differences in PS I/PS II stoichiometries. Cells cultured under different spectral conditions had various PS I/PS II molar ratios when estimated by oxidation-reduction difference absorption spectra of P700 (for PS I) and Cyt b-559 (for PS II) with thylakoid membranes. The fluorescence emission spectra under the Chl a excitation at 435 nm were resolved into several component bands using curve-fitting methods and the relative band area between PS II (F685 and F695) and PS I (F710 or F720) emissions was compared with the PS I/PS II stoichiometries of the various cell types. The results indicated that the PS I/PS II fluorescence ratios correlated closely with photosystem stoichiometries both in Synechocystis sp. PCC 6714 and in C. reinhardtii grown under different light regimes. Furthermore, the correlation between the PS I/PS II fluorescence ratios and the photosystem stoichiometries is also applicable to vascular plants.     

Connection between the rate of cooling and fluorescence properties at 77 K or isolated chloroplasts.

Cooling of chloroplasts to--196 degrees C can under certain circumstances lead to an erroneous analysis of energy distribution. After minimizing influences of sample geometry and effects of plastid concentration it is shown that externally induced membrane change leads to an increase in the ratio F740/F687 of the fluorescence emission spectrum. Similar alterations can be observed by variation of the rate of cooling the plastids to 77 K, expecially if whole chloroplasts are used. The differences in emission ratios are indicative also of changes in initial energy distribution between the photosystems, given here by the value alphaN. This is inferred from experiments with either osmotically induced thylakoid disturbances or those effected through a slow cooling process. The circumstances and the significance of these observations are discussed.     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K II. ATP-dependent quenching

In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{min}$) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F₇₃₅) and a decrease in PS II fluorescence (F₆₉₅) were observed in the low-temperature spectra. The change in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (F_v) and initial (F₀) fluorescence at 690 nm and an increase in F₀ at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.     

Energy distribution in the photochemical apparatus of Porphyridium cruentum: Picosecond fluorescence spectroscopy of cells in state 1 and state 2 at 77 K

Excitation energy distribution in Porphyridium cruentum in state 1 and state 2 was investigated by time resolved 77 K fluorescence emission spectroscopy. The fluorescence rise times of phycoerythrin, phycocyanin and allophycocyanin (in cells in state 1 and state 2) were very similar in contrast to the emission from chlorophyll a (Chl a) associated with the two photosystems. In state 2 photosystem II (PSII) Chl a fluorescence emission rose faster than the PSI Chl a emission and decayed more rapidly, and the converse was observed in state 1. These kinetic data support the concept of increased energy transfer from PSII Chl a to PSI Chl a in state 2 in P. cruentum.Abbreviations APC allophycocyanin - Chl a chlorophyll a - PSII photosystem II - PC phycocyanin - PE phycoerythrin