Fluorescence properties of chlorophyll a and b monomolecular films at the air-water interface

The fluorescence properties of chlorophyll $\text{a}$ and $\text{b}$ monomolecular films at the air-water interface were measured by a high sensitivity fluorophotometer using the photon-counting method. The fluorescence intensity of chlorophyll molecules in monomolecular films in the absence of any diluents did not decrease simply with the mean distance of chlorophyll molecules. Over the range of the mean distances from 27 to 21 Å, three fluorescence components (peaks at 685, 695 and 715 nm) of chlorophyll $\text{a}$ were observed. In the case of chlorophyll $\text{b}$, two fluorescence components (peaks at 667 and 685 nm) were observed over the range of the mean distances from 34 to 24 Å. When the mean distance was 18 Å, the short wavelength component of chlorophyll $\text{b}$ disappeared, and only the long wavelength component was observed.     

Effects of chlorophyll availability on phycobilisomes in Synechocystis sp. PCC 6803

Inactivation of the chlL gene in Synechocystis sp. PCC 6803 resulted in negligible chlorophyll content when the mutant was grown in darkness. Upon phycocyanin excitation at 580 nm, the 77K fluorescence spectrum of dark-grown cells showed three peaks at 648 nm, 665 nm, and 685 nm, this last being the largest. This reflects the functional presence of major components of phycobilisomes, including phycocyanin, allophycocyanin, and the terminal emitter, and efficient energy transfer between these components. As expected, no fluorescence emission peaks corresponding to chlorophyll in the photosystems were observed. Intact phycobilisomes could be isolated from the dark-grown chlL-deletion mutant. However, the phycobilisomes had a lower efficiency of energy transfer than did those isolated from the light-grown mutant, probably because of a decreased phycobilisome stability in the absence of chlorophyll. Exposing the dark-grown chlL-deletion mutant to light triggered the biosynthesis of chlorophyll. For the first 6 h in the light, upon phycocyanin excitation at 580 nm, the 77K fluorescence emission spectrum of greening cells was identical to that of dark-grown cells that lacked significant amounts of chlorophyll. With increased chlorophyll synthesis, gradual energy transfer from phycobilisomes to the two photosystems can be demonstrated.     

Energy transfer by chlorophyll beta in detergent micelles.

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyll beta-containing detergent micelles with Triton X-100 were studied in a concentration range of c equal to 0.4 muM-0.6mM chlorophyll beta and cd equal to 0.4-7.0 mM Triton X-100. The concentration-dependent depolarization obeys F?rster's theory of depolarization of fluorescence with a transfer distance parameter R0 equal to 43 plus or minus 2 A. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yield n/n0 equal to 1/[1+(c/c1/2)-2] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149-161). With increasing chlorophyll beta concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyll beta molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyll beta yields chlorophyll beta homotransfer may be about 3-26% of the homotransfer chlorophyll alpha yields chlorophyll-alpha depending on the ratio of their concentrations.     

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis.

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectively) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 mumol O2/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (-196 degrees C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O2/einstein (605 nm), with a lesser change in the Vmax values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectiely) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 μmol O₂/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (?196°C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O₂/einstein (605 nm), with a lesser change in the V_max values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins, pigment system II particles and chlorophyll a in diethylether solution by the curve-fitting method.

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25 degrees C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol 49, 421--429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm. The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0--2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6--4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively. Absorption spectra at 25 degrees C and at --196 degrees C of the water-soluble chlorophyll proteins were compared by the curve-fitting methods. The component bands at --196 degrees C were blue-shifted by 0.8--4.1 nm and narrower in half widths as compared to those at 25 degrees C.     

Fluorescence properties of the envelope membranes from spinach chloroplasts. Detection of protochlorophyllide

At 77 K, under excitation at 440 nm, two major fluorescence emission peaks were observed in envelope membranes from spinach chloroplasts at 636 and 680 nm. A narrow range of wavelengths around 440 nm and a wider range of wavelengths between 390 and 440 nm, respectively, were responsible for excitation of the 636 and 680 nm fluorescence emissions which, in marked contrast with thylakoid fluorescence emission, were devoid of any exciting components between 460 and 500 nm. In acetonic extract of envelope membranes, two fluorescence emission peaks were observed at 635 and 675 nm. After extraction of the acetonic solution by nonpolar solvents (petroleum ether or hexane), the 675 nm fluorescence emission was partitioned between the polar and nonpolar phases whereas the 635 nm fluorescence emission was solely recovered in the polar phase. All together, the results obtained suggest that envelope membranes contain low amounts of pigments having the absorption and fluorescence spectroscopic properties, together with the behavior in polar/nonpolar solvents, of protochlorophyllide and chlorophyllide. In addition, modulation of the level of fluorescence at 636 and 680 nm could be obtained by addition of NADPH to envelope membranes under illumination. The presence of protochlorophyllide in chloroplast envelope membranes together with its possible photoconversion into chlorophyllide could have major implication for the understanding of chlorophyll biosynthesis in mature chloroplasts.     

Fluorescence and energy transfer in phycobiliprotein-containing algae at low temperature

Fluorescence emission spectra of Anacystis nidulans, Porphyridium cruentum and Cyanidium caldarium, three phycobiliprotein-containing algae, were measured at temperatures between 4 and 120 K in the absence and in the presence of quinones as quenchers of chlorophyll fluorescence. In all species three major emission bands were observed in the chlorophyll a region, near 685 nm (F-685), 695 nm (F-695) and between 710 and 730 nm. Additional bands were observed at shorter wavelengths; these were preferentially excited by light absorbed by the phycobiliproteins and are presumably due to phycocyanins and allophycocyanins.

The amplitudes of F-685, F-695 and the long-wave emission showed a distinct increase upon cooling. For F-685 and F-695 the temperature dependence was similar to that earlier observed with spinach chloroplasts, for the long-wave emission it appeared to depend on the location of the emission bands, which was different for different species. All three bands were strongly quenched by quinones. These and other data suggest that the origin of these bands is the same as in higher plants, and that the fluorescence increase upon cooling can be explained by a lowering of the efficiency of energy transfer between chlorophyll molecules. It is concluded that at most a small percentage of the emission at 685 nm can be ascribed to allophycocyanin B, and that the efficiency of energy transfer between allophycocyanin B and chlorophyll a probably exceeds 99% both at 77 and 4 K. Experiments with isolated phycobilisomes suggest that energy transfer from allophycocyanin to allophycocyanin B occurs with an efficiency of about 90% at low temperature.

The effect of quenchers can be understood by the assumption that the quenching is caused by the formation of non-fluorescent traps in the bulk chlorophyll. Of three quinones tested, the strongest quenching was observed with dibromothymoquinone, which quenched F-685, F-695 and the long-wave emission approximately equally. Menadione and 1,4-naphthoquinone, however, preferentially quenched the long-wave bands, indicating a stronger interaction with Photosystem I than with Photosystem II chlorophylls.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga

The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by pico-second laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a. A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp --At1/2 transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature.

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715--740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50--4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the light-harvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (Fv) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm. From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emitting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

大麦突变种Chlorina-f 2类囊体膜的叶绿素蛋白复合体

当突变种大麦Chlorina-f 2的类囊体膜在SDS/叶绿素的重量比为10:1,叶绿素的浓度为0.5mg/ml的条件下增溶,并在SDS-聚丙烯酰胺凝胶电泳中进行分离时,共出现4条含叶绿素的带。按电泳迁移率的增加,这些带分别是CP Ⅰ,CPa 1,CPa 2和FC。光谱测定表明CP Ⅰ为混有少量光系统Ⅱ??成分的光系统Ⅰ反应中心复合体,CPa 2为光系统Ⅱ反应中心复合体,CPa 2为光系统Ⅱ内周天线复合体。属于光系统Ⅰ的CP Ⅰ的叶绿素含量占总叶绿素的45.6%,而属于光系统Ⅱ的CPa Ⅰ和CPa 2的叶绿素之和则占总叶绿素的43.2%。可见在缺b大麦中,两个都失缺其外周天线的光系统的叶绿素含量是基本相等的。这和光合作用中两个光反应相互串联的理论是完全一致的。     

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons,caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast.     

Monomolecular organization of the main tetraether lipid from Thermoplasma acidophilum at the water-air interface

The monomolecular organization of the main tetraether phospholipid from the archaeon Thermoplasma acidophilum was studied by means of a Langmuir film balance integrated into a fluorescence microscope. After transfer to solid surfaces at different pressures the films were further investigated by ellipsometry, small angle X-ray scattering and atomic force microscopy. In order to complete former results about the main tetraether phospholipid of T. acidophilum [Strobl, C., Six, L., Heckmann, K., Henkel, B., Ring, K., 1985. Z. Naturforsch. 40c, 219-222], the thickness and the two-dimensional organization of the monomolecular films were investigated. Two mean heights values were determined, one of 1.5-1.8 nm and another one of 4-5 nm, indicative for two different molecular arrangements. The former one is interpreted as a 'horseshoe' organization with two polar endings in the aqueous subphase, whereas the latter appears to represent the upright population of molecules with one polar end in the subphase and the other one in the air. In freshly spread and compressed films small domains of the upright lipid population are initially observed, which enlarge with increasing pressure. These domains are no longer existent after 12 h of spreading without compression.     

Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of Photosystem I fluorescence

The time-resolved fluorescence emission and excitation spectra of Chlorella vulgaris cells have been measured by single-photon timing with picosecond resolution. In a three-exponential analysis the time-resolved excitation spectra recorded at 685 and 706 nm emission wavelength with closed PS II reaction centers show large variations of the preexponential factors of the different decay components as a function of wavelength. At λ_em = 685 nm the major contribution to the fluorescence decay originates from two components with life-times of 2.1–2.4 and 1.2–1.3 ns. A short-lived component with life-times of 0.1–0.16 ns of relatively small amplitude is also found. When the emission is detected at 706 nm, the short-lived component with a life-time of less than 0.1 ns predominates. Time-resolved emission spectra using λ_exc = 630 or λ_exc = 652 nm show a spectral peak of the two longer-lived components at about 680–685 nm, whereas the fast component is red-shifted as compared to the others and shows a maximum at about 690 nm. The emission spectrum observed upon excitation at 696 nm with closed PS II reaction centers shows a large increase in the amplitude of the fast component with a lifetime of 80–100 ps as compared to that at 630 nm excitation. At almost open Photosystem II (PS II) reaction centers (F₀), the life-time of the fast component decreased from 150–160 ps at 682 nm to less than 100 ps at 720 nm emission wavelength. We conclude that at least two pigment pools contribute to the fast component. One is attributed to PS II and the other to Photosystem I (PS I). They have life-times of approx. 180 ps and 80 ps, respectively. The 80 ps (PS I) contribution has a spectral maximum slightly below 700 nm, whereas the 180 ps (PS II) spectrum peaks at 680–685 nm. The spectra of the middle decay component τ_m and its sensitivity to inhibitors of PS II suggest that this component is not preferentially related to LHC II but arises mainly from Chl a pigments probably associated with a second type of PS II centers. The amplitudes of the fast (180 ps, PS II) component and the long-lived decay show an opposite dependence on the state of the PS II centers and confirm our earlier conclusion that the contribution of PS II to the fast component probably disappears at the F_max state (Haehnel W., Holzwarth, A.R. and Wendler, J. (1983) Photochem. Photobiol. 34, 435–443). Our data are discussed in terms of α,β-heterogeneity in PS II centers.     

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy.

The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a.     

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons, caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715–740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50-4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the lightharvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (F_v) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm.From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emittting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

A retrieval algorithm to evaluate the Photosystem I and Photosystem II spectral contributions to leaf chlorophyll fluorescence at physiological temperatures

A new computational procedure to resolve the contribution of Photosystem I (PSI) and Photosystem II (PSII) to the leaf chlorophyll fluorescence emission spectra at room temperature has been developed. It is based on the Principal Component Analysis (PCA) of the leaf fluorescence emission spectra measured during the OI photochemical phase of fluorescence induction kinetics. During this phase, we can assume that only two spectral components are present, one of which is constant (PSI) and the other variable in intensity (PSII). Application of the PCA method to the measured fluorescence emission spectra of Ficus benjamina L. evidences that the temporal variation in the spectra can be ascribed to a single spectral component (the first principal component extracted by PCA), which can be considered to be a good approximation of the PSII fluorescence emission spectrum. The PSI fluorescence emission spectrum was deduced by difference between measured spectra and the first principal component. A single-band spectrum for the PSI fluorescence emission, peaked at about 735?nm, and a 2-band spectrum with maxima at 685 and 740?nm for the PSII were obtained. A linear combination of only these two spectral shapes produced a good fit for any measured emission spectrum of the leaf under investigation and can be used to obtain the fluorescence emission contributions of photosystems under different conditions. With the use of our approach, the dynamics of energy distribution between the two photosystems, such as state transition, can be monitored in vivo, directly at physiological temperatures. Separation of the PSI and PSII emission components can improve the understanding of the fluorescence signal changes induced by environmental factors or stress conditions on plants.