Migration of Electronic Energy from Chlorophyll b to Chlorophyll a in Solutions

Absorption, emission, and fluorescence excitation spectra of pure solutions of chlorophyll a (Chl a) and chlorophyll b (Chl b) in diethyl ether and of equimolecular mixed solutions of the two pigments, were determined at room temperature as functions of concentration (in the range from 5 × 10^-6 M to 4 × 10^-3 M) and of wavelength of the exciting light (in the regions 380-465 and 550-650 nm). The efficiency of energy transfer from Chl b to Chl a, derived from these data, was found to depend on the wavelength of exciting light. Furthermore, the transfer efficiency calculated from sensitization of Chl a fluorescence by Chl b was substantially smaller than that calculated from quenching of Chl b fluorescence by Chl a. Both these effects are tentatively explained as evidence of superposition of a “fast” energy transfer (taking place before the Boltzmann distribution of vibrational energy had been reached) upon the “delayed” transfer, which takes place after vibrational equilibration. The first-named mechanism is made possible by overlapping of the absorption bands of the two pigments; the second, by overlapping of the emission band of Chl b and the absorption band of Chl a. The first mechanism can lead to repeated transfer of excitation energy between pigment molecules, the second only to a one-time transfer from the donor to the acceptor. Both mechanisms could be of the same, second-order type, with the transfer rate proportional to r^-6. An alternative is for the fast mechanism to be of the first order, with the transfer rate proportional to r^-3, but spectroscopic evidence seems to make this alternative less probable.     

快速叶绿素荧光诱导动力学分析在光合作用研究中的应用

JIP-测定(JIP-test)是以生物膜能量流动为基础建立的分析方法.利用该方法可以获得有关光系统Ⅱ的大量信息.文章介绍了快速叶绿素荧光诱导动力学曲线的定义、数据分析方法及相关参数的意义,并举例说明如何利用该方法分析不同环境条件对光合机构主要是PSⅡ的供体侧、受体侧及PSⅡ反应中心的影响.     

Chlorophyll fluorescence characteristics associated with hydration level in pea cotyledons

In order to study the effects of desiccation on a photosynthetic system, light harvesting and light-induced electron transport processes were examined in pea cotyledons at various moisture levels, using in vivo fluorescence excitation spectra and fluorescence induction kinetics. Water sorption isotherms yielded thermodynamic data that suggested very strong water binding between 4 to 11% water, intermediate sorption between water contents of 13 to 22%, and very weak binding at moisture contents between 24 to 32%. The fluorescence properties of the tissue changed with the moisture contents, and these changes correlated generally with the three regions of water binding. Peak fluorescence and fluorescence yield remained at low levels when water content was limited to the tightly bound regions, below 12%. Several new peaks appeared in the chlorophyll a excitation spectrum and both peak fluorescence and fluorescence yield increased at intermediate water-binding levels (12-22%). At moisture contents where water is weakly bound (>24%), peak fluorescence and fluorescence yield were maximum and the fluorescence excitation spectrum was unchanging with further increases in water content.

The state of water is an important component in the energy transfer and electron transport system. At hydration levels where water is most tightly bound, energy transfer from pigments is limited and electron transport is blocked. At intermediate water binding levels, energy transfer and electron transport increase and, in the region of weak water binding, energy transfer and electron transport are maximized.

     

Spectral properties of the CP43-deletion mutant of Synechocystis sp. PCC 6803

Spectral properties, particularly fluorescence spectra and their time-dependent behavior, were investigated for a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the 43 kDa chlorophyll-protein (CP43, PsbC). Lack of CP43 was confirmed by a size shift of the corresponding gene and by Western blotting. The CP43-deletion mutant grown under heterotrophic conditions accumulated a small amount of photosystem (PS) II, but virtually no PS II fluorescence was observed. A 686-nm fluorescence band was clearly observed by phycocyanin excitation, coming from the terminal pigments of phycobilisomes. In contrast, no PS I fluorescence was detected by phycocyanin excitation when accumulation of PS II components was not proved by a fluorescence excitation spectrum, indicating that energy transfer to PS I chlorophyll a was mediated by PS II chlorophyll a. Direct connection of phycobilisomes with PS I was not suggested. Based on these fluorescence properties, the energy flow in the CP43-deletion mutant cells is discussed.     

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Excitation energy transfer in the light-harvesting chlorophyll

The “light-harvesting chlorophyll a/b · protein” described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600–700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitation dependence of the fluorescence polarization shows a minimum polarization of 1.9 % at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8 % at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a doublet structure in the chlorophyll b absorption band which suggests an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the S₀→S₁ transition moments of the chlorophyll molecules within the protein.     

Picosecond energy transfer in Porphyridium cruentum and Anacystis nidulans.

Picosecond energy transfer is measured in Anacystis nidulans and Porphyridium cruentum. Fluorescence is sensitized by a 6-ps laser flash, at 530 nm. The time dependence of fluorescence is measured with reference to the laser pulse. Fluorescence is recorded from phycoerythrin (576 nm), R-phycocyanin (640 nm), allophycocyanin (666 nm), Photosystem II chlorophyll (690 nm) and long wave length chlorophyll (715 nm). Energy transfer measurements are made at 37 degrees C, 23 degrees C, and 0 degrees C, and 77 degrees K. It is shown that the rate of energy transfer can be varied with temperature. In both A. nidulans and P. cruentum there is a sequential transfer of excitation energy from phycoerythrin to phycocyanin to allophycocyan to Photosystem II chlorophyll fluorescence. The long wavelength chlorophyll fluorescence at 715 nm, however, does not always follow a sequential transfer of excitation energy. Depending on the temperature, fluorescence at 715 nm can precede fluorescence from phycocyanin.     

In vivo Chlorophyll Fluorescence Monitoring with Solid State Photosensors

Solid state photosensors with their many advantages have not been utilized for chlorophyll fluorescence research. One reason is their reputed low photosensitivity compared to photomultiplier tubes. A photodarlington sensor has a relatively low photosensitivity and a nonlinear current output versus light intensity. However, when incorporated into an apparatus described in this paper it can respond to fluorescence signals from leaves using light excitation as low as 500 μW/cm². The sensitivity of another solid state device (photovoltaic photodiode) was compared to five photomultipliers using a monochromator-microphotometer testing apparatus. This solid state sensor proved to have equal or greater sensitivity to chlorophyll fluorescence. An apparatus incorporating the photovoltaic photodiode is discussed.     

Chlorophyll a fluorescence measurements of isolated spinach thylakoids obtained by using single-laser-based flow cytometry

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.     

Thermal dissipation of light energy is regulated differently and by different mechanisms in lichens and higher plants

Modulated chlorophyll fluorescence was used to compare dissipation of light energy as heat in photosystem II of homoiohydric and poikilohydric photosynthetic organisms which were either hydrated or dehydrated. In hydrated chlorolichens with an alga as the photobiont, fluorescence quenching revealed a dominant mechanism of energy dissipation which was based on a protonation reaction when zeaxanthin was present. CO2 was effective as a weak protonating agent and actinic light was not necessary. In a hydrated cyanobacterial lichen, protonation by CO2 was ineffective to initiate energy dissipation. This was also true for leaves of higher plants. Thus, regulation of zeaxanthin-dependent energy dissipation by protonation was different in leaves and in chlorolichens. A mechanism of energy dissipation different from that based on zeaxanthin became apparent on dehydration of both lichens and leaves. Quenching of maximum or Fm fluorescence increased strongly during dehydration. In lichens, this was also true for so-called basal or Fo fluorescence. In contrast to zeaxanthin-dependent quenching, dehydration-induced quenching could not be inhibited by dithiothreitol. Both zeaxanthin-dependent and dehydration-induced quenching cooperated in chlorolichens to increase thermal dissipation of light energy if desiccation occurred in the light. In cyanolichens, which do not possess a zeaxanthin cycle, only desiccation-induced thermal energy dissipation was active in the dry state. Fluorescence emission spectra of chlorolichens revealed stronger desiccation-induced suppression of 685-nm fluorescence than of 720-nm fluorescence. In agreement with earlier reports of , fluorescence excitation data showed that desiccation reduced flow of excitation energy from chlorophyll b of the light harvesting complex II to emitting centres more than flow from chlorophyll a of core pigments. The data are discussed in relation to regulation and localization of thermal energy dissipation mechanisms. It is concluded that desiccation-induced fluorescence quenching of lichens results from the reversible conversion of energy-conserving to energy-dissipating photosystem II core complexes.     

Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi

We have investigated the process of intermolecular excitation energy transfer and the relative orientation of the chlorophyll molecules in the unicellular green alga Chlamydomonas reinhardi. The principal experiments involved in vivo measurements of the fluorescence polarization as a function of the exciting-light wavelength in the presence and in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We found that as the fluorescence lifetime increases upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea that the degree of fluorescence polarization decreases over the excitation region from 600 to 660 nm. This result, we argue, implies that a Förster mechanism of excitation energy transfer is involved for Photosystem II chlorophyll molecules absorbing primarily below 660 nm. We must add that our results do not exclude the possibility of a delocalized transfer process from being involved as well. Fluorescence polarization measurements using chloroplast fragments are also discussed in terms of a Förster transfer mechanism. As the excitation wavelength approaches 670 nm the fluorescence polarization is nearly constant upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.Experiments performed using either vertically or horizontally polarized exciting light show that the fluorescence polarization increases as the exciting light wavelength increases from 650 to 673 nm. This suggests the possibility that chlorophyll molecules absorbing at longer wavelengths have a higher degree of relative order. Furthermore, these studies imply that chlorophyll molecules exist in discrete groups that are characterized by different absorption maxima and by different degrees of the fluorescence polarization. In view of these results we discuss different models for the Photosystem II antenna system and energy transfer between different groups of optically distinguishable chlorophyll molecules.     

The fluorescence spectra of red algae and the transfer of energy from phycoerythrin to phycocyanin and chlorophyll

1. The fluorescence spectra of the alga Porphyridium have been recorded as energy distribution curves for eleven different incident wave lengths of monochromatic incident light between wave lengths 405 and 546 mµ. 2. In these spectra chlorophyll fluorescence predominates when the incident light is in the blue part of the spectrum which is strongly absorbed by chlorophyll. 3. For blue-green and green light the spectrum excited in Porphyridium contains in addition to chlorophyll fluorescence, the fluorescence bands characteristic of phycoerythrin and of phycocyanin. 4. From these spectra the approximate curves for the fluorescence of the individual pigments phycoerythrin, phycocyanin, and chlorophyll in the living material have been derived and the relative intensity of each of them has been obtained for each of the eleven incident wave lengths. 5. The effectiveness spectrum for the excitation of the fluorescence of these three pigments in vivo has been plotted. 6. From comparisons of the effectiveness spectrum for the excitation of each of these pigments it appears that both phycocyanin and chlorophyll receive energy from light which is absorbed by phycoerythrin. 7. It is suggested that phycocyanin may be an intermediate in the resonance transfer of energy from phycoerythrin to chlorophyll. 8. Since phycoerythrin and phycocyanin transfer energy to chlorophyll, it appears probable that chlorophyll plays a specific chemical role in photosynthesis in addition to acting as a light absorber.     

Excitation energy transfer in the light-harvesting chlorophyll a/b.protein

The "light-harvesting chlorophyll a/b.protein" described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600-700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitiation dependence of the fluorescence polarization shows a minimum polarization of 1.9% at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8% at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a double structure in the chlorophyll b absorption band which suggest an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the So leads to S1 transition moments of the chlorophyll molecules within the protein.     

用体内叶绿素a荧光诱导动力学鉴定番茄的抗冷性

研究了冷害温度对具有不同抗冷性品种的番茄叶片的体内叶绿素a荧光诱导动力曲线的影响。实验结果指出,在低温处理(8℃,5℃,2℃下,暗中24小时)后,番茄叶片的体内叶绿素a荧光诱导动力学曲线有了明显的改变,Fv／Fo值、Rfd值降低了,光系统II原初光能转换效率和潜在的光合活力均受到抑制。我们在苗期和开花期得到的实验结果均表明,在番茄叶片的叶绿素a荧光诱导动力学曲线和这些荧光参数改变的程度与该品种的已知抗冷性之间呈现较好的相关性。我们认为,体内叶绿素a荧光诱导动力学方法是鉴定番茄抗冷性的一个快速、灵敏和可靠的方法,并可用于其他绿色植物的抗冷性鉴定中。     

基于叶绿素荧光成像及荧光参数分布特征的叶片光合异质性定量分析

植物叶片光合作用具有高度的空间异质性,叶绿素荧光成像技术为叶片光合异质性的研究提供了便利,但叶片光合异质性的定量分析并没有得到广泛应用。本文利用Imaging PAM叶绿素荧光成像系统,获得中亚热带地区越冬期小叶榕(Ficus microcarpa)阳生叶和阴生叶的叶绿素荧光参数图像,并利用仪器的分析软件对其进行分析,定量比较了阳生叶和阴生叶的光合异质性特征。研究发现：越冬期小叶榕阳生叶的光合异质性和光抑制程度明显高于阴生叶,变异系数可作为光合异质性的定量指标。低温强光导致阳生叶坏死率(P_LN)达4.30%,并有53.30%的区域处于严重光抑制(0v/F_m<0.627),但仍有42.27%的区域仅为轻度光抑制(0.627≤F_v/F_m<0.800)。而低温弱光并未造成阴生叶坏死和严重光抑制。通过对光系统Ⅱ(PSⅡ)的实际光合效率(Y(Ⅱ))、调节性能量耗散的量子产额(Y(NPQ))和非调节性能量耗散的量子产额(Y(NO))荧光参数异质性的定量分析表明,阳生叶...     

Low-Temperature Fluorescence Excitation Spectra for Long-Wavelength Emission as a Function of Greening in Euglena gracilis and Chlorophyll A Concentration In Vitro: A Mathematical Model to Describe Both Systems

A systematic study was made of the spectrum for exciting long-wave-length fluorescence (at 77°K) during the first 100 hr of greening in Euglena gracilis. A band at 705-710 nm is observable after cells have been greening in light for 30 hr. The ratio of the 705-nm to the 675-nm peak increases during greening, reaching a maximum value at 85 hr, then declining. With concentrated solutions of chlorophyll a, fluorescence excitation spectra are similar to those observed in vivo. The ratio of aggregate to monomer bands increases with concentration of chlorophyll, reaching a maximum value in ethanol and in pyridine at about 3 × 10^-2 M and 6 × 10^-2 M respectively, then declining. Several model systems were analyzed. It is shown that the band observed in solution with maximum at 705-710 nm is not an artifact of the fluorescence apparatus; it does not arise from undissolved chlorophyll; it does not arise from a fluorescent or nonfluorescent impurity; it does not arise solely from light absorption by a dimer or larger aggregate of chlorophyll. Agreement is obtained between the experimental observations and the results of a mathematical model by including terms for the efficiency of energy transfer from monomeric to dimeric chlorophyll, as well as for the formation of dimers by an equilibrium reaction.     

Spectral analysis on origination of the bands at 437 nm and 475.5 nm of chlorophyll fluorescence excitation spectrum in Arabidopsis chloroplasts

Chlorophyll fluorescence has been often used as an intrinsic optical molecular probe to study photosynthesis. In this study, the origin of bands at 437 and 475.5 nm in the chlorophyll fluorescence excitation spectrum for emission at 685 nm in Arabidopsis chloroplasts was investigated using various optical analysis methods. The results revealed that this fluorescence excitation spectrum was related to the absorption characteristics of pigment molecules in PSII complexes. Moreover, the excitation band centred at 475.5 nm had a blue shift, but the excitation band at 437 nm changed relatively less due to induction of non‐photochemical quenching (NPQ). Furthermore, fluorescence emission spectra showed that this blue shift occurred when excitation energy transfer from both chlorophyll b (Chl b) and carotenoids (Cars) to chlorophyll a (Chl a) was blocked. These results demonstrate that the excitation band at 437 nm was mainly contributed by Chl a, while the excitation band at 475.5 nm was mainly contributed by Chl b and Cars. The chlorophyll fluorescence excitation spectrum, therefore, could serve as a useful tool to describe specific characteristics of light absorption and energy transfer between light‐harvesting pigments. Copyright © 2015 John Wiley & Sons, Ltd.     

黄化油菜突变体Cr3529子叶类囊体膜光谱性质研究

以发育10d的黄化油菜突变体为材料,分析了突变体油菜子叶类囊体膜的色素含量、室温吸收光谱、叶绿素荧光发射和激发光谱以及蛋白内源荧光光谱的变化。数据显示：与野生型相比,突变体油菜子叶类囊体膜的光合色素Chl α和Chl b含量均减少．但Chl α／b比值升高;突变体油菜子叶类囊体膜叶绿素捕光能力和受激发能力均下降,且较依赖于Chl α捕光并将光能激发传递给PSⅡ反应中心;突变体油菜子叶类囊体膜的蛋白内源荧光也明显异于野生型。进一步表明突变体油菜子叶类囊体膜蛋白组成发生了改变。     

Chlorophyll: an efficient detector of electronically excited species in biochemical systems

Micelle-solubilized chlorophyll efficiently detects electronically excited species generated in enzymatic systems. In most, if not all, systems the chemiexcited species is formed in the triplet state; chlorophyll fluorescence is observed as result of energy transfer. Red emission can also be elicited from chlorophyll in chloroplasts or bound to microsomes.     

DIFFERENCES BETWEEN IN VIVO ABSORPTION AND FLUORESCENCE EXCITATION SPECTRA IN NATURAL SAMPLES OF PHYTOPLANKTON

The fluorescence excitation spectrum of live phytoplankton cells represents the portion of light absorbed that has been effectively transferred to chlorophyll a of photosystem II, whereas light absorbed by photoprotective pigments will not lead to fluorescence. Therefore, the in vivo fluorescence excitation spectrum of phytoplankton has been used as a proxy for the action spectrum of phytoplankton in computations of primary production in the ocean. The distribution of chlorophyll a between photosystems, as well as variations in the pathway of energy inside the photosynthetic membrane, can also influence the fluorescence excitation spectrum. In this study, we investigated the contribution of photoprotective pigments to the differences found between in vivo absorption and fluorescence excitation spectra of phytoplankton measured during two cruises: one from Las Islas Canarias to Nova Scotia and another in the Labrador Sea. A comparison of normalized fluorescence excitation and absorption spectra showed high variability in the difference between absorption and fluorescence in the blue region of the spectrum for samples from the two cruises. This difference was not entirely correlated with the concentration of photoprotective carotenoids. In this paper, results are interpreted in terms of differences in pigment composition and known patterns of energy distribution in the photosystems of different algal groups.