High light-induced changes of 77 K fluorescence emission of pea thylakoid membranes with altered membrane fluidity

The effect of lipid phase order of isolated thylakoid membranes on fluorescent characteristics of both photosystems during illumination with high light intensity at 22 degrees C and 4 degrees C was investigated. For artificial modification of membrane fluidity two membrane perturbing agents were applied-cholesterol and benzyl alcohol. 77 K fluorescence emission and excitation spectra of control, cholesterol- and benzyl alcohol-treated thylakoid membranes were analysed in order to determine the high light-induced changes of emission bands attributed to different chlorophyll-protein complexes-F 735, emitted by photosystem I-light-harvesting complex I; and F 685 and F 695, emitted by photosystem II-light-harvesting complex II. Analysis of emission bands showed that high light treatment leads to a decrease of the area of band at 695 nm and a concomitant increase of intensity of the band at 735 nm. The involvement of different pigment pools (chlorophyll a and chlorophyll b) in the energy supply of both photosystems before and after photoinhibitory treatment was estimated on the basis of excitation fluorescence spectra. The dependence of the ratios F 735/F 685 and the band areas at 685 and 695 nm on the illumination time was studied at both temperatures. Data presented indicate that cholesterol incorporation stabilized the intersystem structure in respect to light-induced changes of fluorescence emission of PSI and PSII. It was shown that the effect of fluid properties of thylakoid membranes on the 77 K fluorescence characteristics of main pigment protein complexes of pea thyalkoid membranes depends on the temperature during high light treatment.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

CHLOROPHYLL a FLUORESCENCE OF GONYAULAX POLYEDRA GROWN ON A LIGHT-DARK CYCLE AND AFTER TRANSFER TO CONSTANT LIGHT

Abstract— The chlorophyll a fluorescence properties of Gonyaulax polyedra cells before and after transfer from a lightdark cycle (LD) to constant dim light (LL) were investigated. The latter display a faster fluorescence transient from the level ‘I’ (intermediary peak) to ‘D’ (dip) to ‘P’ (peak) than the former (3 s as compared to 10 s), and a different pattern of decline in fluorescence from ‘I’ to ‘D’ and from ‘P’ to the steady state level with no clearly separable second wave of slow fluorescence change, referred to as ‘s' (quasi steady state)→‘M’ (maximum) →‘T’ (terminal steady state). The above differences are constant features of cells in LD and LL, and are not dependent on the time of day. They are interpreted as evidence for a greater ratio of photosystem II/photosystem I activity in cells in LL. After an initial photoadaptive response following transfer from LD to LL, the cell absorbance at room temperature and fluorescence emission spectra at 77 K for cells in LL and LD are comparable. The major emission peak is at 685–688 nm (from an antenna Chl a 680, perhaps Chl a-c complex), but, unlike higher plants and other algae, the emission bands at 696–698 nm (from Chl a_II complex, Chl a 685, close to reaction center II) and 710–720 nm (from Chl a₁, complexes, Chl a 695, close to reaction center I) are very minor and could be observed only in the fluorescence emission difference spectra of LL minus LD cells and in the ratio spectra of DCMU-treated to non-treated cells. Comparison of emission spectra of cells in LL and LD suggested that, in LL, there is a slightly greater net excitation energy transfer from the light-harvesting peridinin-Chl a (Chl a 670) complex, fluorescing at 675 nm, to the other antenna chlorophyll a complex fluorescing at 685–688 nm, and from the Chl a., complex to the reaction center II. Comparison of excitation spectra of fluorescence of LL and LD cells, in the presence of DCMU, confirmed that cells in LL transfer energy more extensively from the peridinin-Chl a complex to other Chl a complexes than do cells in LD.     

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.     

PHOTOSYNTHETIC ACTION SPECTRA AND LIGHT-HARVESTING IN GRIFFITHSIA MONILIS (RHODOPHYTA)

Abstract— In cells of the red alga Griffithsia monilis the action spectrum of photosynthetic oxygen production at low light intensity shows that the phycobilins (including allophycocyanin) are the major light-harvesting pigments. As the light intensity is increased carotenoids and chlorophyll a contribute proportionately more to the spectrum, since the phycobilin activity becomes light-saturated. When action spectra are performed against a background light of various monochromatic wavelengths it can be shown that chlorophyll a increases in its light-harvesting activity. Nevertheless light absorbed at a single wavelength (487 nm) by phycoerythrin (and possibly a carotenoid) still shows the highest photosynthetic activity. Fluorescence measurements at 77K indicate that a chlorophyll a fluorescence is small and that the amount of chlorophyll a _ll (f 693) is very low. A model is proposed in which the phycobilins, in phycobilisomes, pass on absorbed light energy to either photosystem, whereas light absorbed by chlorophyll is passed on mainly to photosystem I.     

SPECTROSCOPIC STUDIES OF CHLOROPHYLL a AGGREGATES FORMED BY AQUEOUS DIMETHYL SULFOXIDE

Characteristic chlorophyll (Chl) a aggregates formed in aqueous dimethyl sulfoxide (DMSO) were investigated spectroscopically. Four chlorophyll forms were found with increasing DMSO concentration; they are called A-672, A-683, A-695 and A-665 according to the wavelengths of their absorption maxima. Transformation occurred only in this order. Reverse transformation could not be realized. A-683 and A-695 were apparently formed by the interaction of Chl a with DMSO in the linear dimer and linear polymer arrangements, respectively. Coordination of the Mg atom with a DMSO O atom and interaction between the S atom of one DMSO molecule and the O atom of an other DMSO molecule should lead to formation of a sandwich-type complex of partially overlapping chlorophyll macrocycles (Chl a-DMSO)_n. A-672 and A-665 were assigned to Chl a micelles and to dissolved monomeric Chl a in DMSO, respectively. Fluorescence spectra showed that the A-683 was highly fluorescent, while the A-695 was less fluorescent. Energy migration within the A-695 form to a trap with a low fluorescence yield might be responsible for this difference in the emission intensity.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

FLUORESCENCE SPECTROSCOPY OF A P700-CHLOROPHYLL-PROTEIN COMPLEX*

Abstract. Chlorophyll-protein complexes enriched in the Photosystem I reaction center chlorophyll (P700) exhibit a fluorescence emission maximum at 696 nm at - 196°C The height of this 696 nm emission relative to the emission at 683 nm from antenna chlorophyll a increases proportionally with the P700 concentration while the total fluorescence yield of the complex decreases. The 696 nm emission could possibly be from an absorbing form of antenna chlorophyll a that may be somewhat enriched along with P700 in Photosystem I fractions. However, evidence resulting from glycerol treatment which appears to decrease the rate of resonance energy transfer between antenna chlorophyll and P700 favors the hypothesis that the emission comes from a photooxidized P700 dimer (Chl⁺-Chl) absorbing near 690 nm. In turn, this fluorescence evidence provides additional support for the model of a P700 dimer involving exciton interaction. Absorption in the wavelength region of 450 nm specifically excites emission at 696 nm from the P700-chlorophyll complex.     

IN VIVO FLUORESCENCE SPECTROSCOPY OF CHLOROPHYLL IN VARIOUS UNRIPE AND RIPE FRUIT

—Low temperature (77 K) fluorescence emission spectra of slices obtained from the peel and various layers of the pericarp were recorded for fruits which remain green or undergo color break during ripening.
Fluorescence emission peaks characteristic of the photosystem II antennae (λ_F 686 nm) and reaction center (λ_F 696 nm), as well as of the photosystem I antenna (λ_F 730-740 nm), were present in the peel and all parts of the green pericarp of ripe kiwi, avocado and cantaloupe, as well as in ripe tomato and tangerine after color break. The pattern of the fluorescence emission spectra of all samples except that of the kiwi fruit was similar to that obtained from green photosynthetic tissue of leaves, indicating a normal organization of the chlorophyll-containing complexes of thylakoidal membranes. This pattern is characterized by a significantly higher emission at 730-740 nm relative to that of the 696 and 686 nm peaks. In contradistinction, the fluorescence emission at 686 and 696 nm was higher than that at 730 nm in the kiwi fruit, indicating a reduction in the size of the photosystem I antenna chlorophyll. In the innermost yellowish layers of the kiwi pericarp, a further loss of this antenna occurred, as well as disorganization of the photosystem II complex. The above conclusions are suggested also by measurements of variable fluorescence kinetics.
The results presented here indicate that fluorescence spectroscopy might be used as a tool for the study of chlorophyll organization during the growth and ripening periods of fruit.     

ASSOCIATION OF CHLOROPHYLL WITH AMIDES ON PLASTICIZED POLYETHYLENE PARTICLES—I. N,N-DIMETH YLM YRISTAMIDE*

Abstract— Model systems have been prepared in which chlorophyll a (Chl a) and N.N-dimethylmyristamide (DMMA) are adsorbed together in various ratios to particles of polyethylene swollen with undecane. The adsorption is performed by equilibrating the particles with methanol-water solutions of increasing water content. Absorption spectra of the coated particles in viscous suspenions show sharp well-marked bands over much of the composition range examined. With the aid of second derivative spectra. the red absorption band has been resolved into three components. at 661.5. 674 and 680 nm. Fluoresccnce spectra have also been resolved into their principal components with some assistance from comparison with spectra of Chl in undecane solution containing DMMA. At room temperature (295 K) the resolvable components are of monomeric Chl at 670 nm. and of associated species at 681 and 725 nm. Fluorescence at 77 Kis of similar intensity but is distributed differently. in favor of longer-wave components. Corresponding to the 295 K components are emission bands at 675, 683–5 and 735 nm. Othcr components appear under certain conditions: at 695–700 nm when the Chl and DMMA conccntrations are both high, and at 705 nm whcn the ratio of DMMA to Chl is low. If DMMA is absent or at low concentration, much of the Chl exists as an aggregated form absorbing near 741 nm and fluorescing weakly near 760 nm at 77 K. Adsorption isotherms indicate some degree of cooperativity in the binding of Chl and DMMA to the particles. The photoreduction of p-dinitrobenzene by hvdrazobenzene. scnsitized by these particles, has been demonstrated     

EXCITATION ENERGY TRANSFER IN THE CRYPTOPHYTES. FLUORESCENCE EXCITATION SPECTRA AND PICOSECOND TIME-RESOLVED EMISSION SPECTRA OF INTACT ALGAE AT 77 K

Excitation spectra of chlorophyll- a (Chl- a ) fluorescence in intact cells of Cryptomonas ovata, Chroomonas pauciplastida and Chroomonas salina were determined at 77 K. For all species the excitation spectra for emission from Chl- a associated with photosystem II (PSII) showed increased contributions by a carotenoid (493 nm) and phycobiliproteins, and decreased contributions by carotenoid (417 nm, 505 nm) and Chl- a (445 nm) as compared to excitation spectra for emission from Chl- a associated with photosystem I (PSI). Excitation spectra of C. salina and C. ovata showed an increased contribution by Chl- c ² to PSII Chl- a fluorescence emission. In all three species the absorbance band positions of Chl- a , as determined from the excitation spectra, were similar to those previously described in green plants. green algae and phycobilisome-containing organisms. Time-resolved 77 K fluorescence emission spectra of C. ovata and C. salina showed successive emission from both phycoerythrin and Chl- c ², PSII Chl- a , and PSI Chl- a. C. pauciplastida showed successive emission from phycocyanin, PSII Chl- a , and PSI Chl- a. Spectral red-shifts with time were observed for the phycobiliprotein peaks in all three species. The fluorescence decay of phycoerythrin in C. ovata and C. salina was faster than that of phycocyanin in C. pauciplastida. The results are discussed in relation to the organization of the antenna pigments of PSII and PSI in the cryptophyte algae.     

POLARIZED PHOTO ACOUSTIC SPECTRA OF PHYCOBILISOMES IN POLYVINYL ALCOHOL FILMS

Abstract— Phycobilisomes from Porphyridium cruentum , suspended in polyvinyl alcohol were found to be highly stable, and had normal absorption and fluorescence spectra. Intact phycobilisomes had a major emission peak at 680 nm, whereas upon partial dissociating the major emission was at 580 nm. Incorporation of phycobilisomes in thin polyvinyl alcohol films facilitated examination by photoacoustic spectroscopy. The photoacoustic spectra had a broad absorption maximum at 545–565 nm (phycoerythrin), which resolved as two peaks (545 and 563 nm) in absorption spectra. Stretching of films resulted in apparent chromophore reorientation in partially dissociated, but not in intact phycobilisomes. Only in dissociated phycobilisomes was observed a differential chromophore orientation at 685 nm by polarized fluorescence, which is attributed to a change in orientation of the terminal phycobilisome pigment relative to phycoerythrin.     

CHANGES IN FLUORESCENCE SPECTRA OF CHLOROPLASTS INDUCED BY A NATURALLY-OCCURRING FACTOR

Abstract— –Fluorescence emission changes (measured at – 196°C) of Ricinus chloroplasts incubated in isolation medium, and of chloroplasts from algae and higher plants incubated in Ricinus leaf extract, are described. Such incubation results in a transformation of the three-banded emission spectrum (F735, F698, F685) into a virtually one-banded spectrum, with maximum at 698 nm. That these changes are a consequence of the conversion (deaggregation) of the form of chlorophyll giving rise to F735 into a form contributing to fluorescence at 698 nm is suggested on the basis of room temperature absorption and low temperature fluorescence excitation studies, made concomitantly with the low temperature emission studies.     

EXCITATION ENERGY MIGRATION IN PHYCOBILISOMES: COMPARISON OF EXPERIMENTAL RESULTS AND THEORETICAL PREDICTIONS

Abstract— Quantum yield and fluorescence polarization determinations on phycobilisomes and their constituent phycobiliproteins show that phycobilisomes are energetically effective macromolecular structures. Energy migration within the phycobilisome to allophycocyanin, the longest wavelength absorbing and emitting phycobiliprotein, was indicated by the predominant allophycocyanin fluorescence emission which was independent of the phycobiliprotein being excited. The high efficiency of the energy migration inside the phycobilisome was reflected by the low polarized fluorescence. Excitation of phycobilisomes in the region of major absorption (500–650 nm) resulted in degrees of fluorescence polarization between +0.02 and –0.02, whereas in isolated phycobiliproteins the values were 2 to 12 times greater. Furthermore, 94–98° of the excitation energy of phycoerythrin was transferred to phycocyanin and allophycocyanin as determined from comparisons of fluorescence spectra of intact and dissociated phycobilisomes. The fluorescence quantum yields of phycobilisomes were about 0.60–0.68, very similar to that of pure allophycocyanin in solution (0.68). Phycobilisomes isolated from Fremyella diplosiphon and Nostoc sp. (blue-gree algae) have respective quantum yields of 0.68 and 0. 65, and those isolated from Porphyridium cruentum (red alga), about 0.60. In Fremyella diplosiphon and Nostoc sp., which showed a striking adaptation to different wavelengths, the phycobilisome quantum yields only varied from 0.68 to 0.67 and from 0.65 to 0. 60, respectively. The mean transfer time, calculated on the basis of experimental results, was about 280 ± 40 ps for transfer of excitation from the phycoerythrin to the phycocyanin layer in phycobilisomes. This time corresponds to the mean number of jumps, about 28, of the excitation in the phycoerythrin layer before it is captured by phycocyanin. These values are in reasonable agreement with the values of 250 ± 30 ps and 25 jumps, calculated on the basis of a phycobilisome model (of Porphyridium cruentum) and Pearlstein's theory of energy migration devised for a three-dimensional photosynthetic unit. It was also shown that Paillotin's theory of energy migration predicts similar values for mean transfer time and mean number of jumps, if one assumes that phycocyanin is a perfect sink for phycoerythrin excitation.     

Changes in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F(O) (basal level of fluorescence yield at transient O) and F(M) (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F(O) to F(M), a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F(M) to F(T) (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII.     

Highly resolved luminescence measurements for traces of 1,3-dibetanato europium(III) and terbium(III) complexes using a microscope laser Raman spectrometer toward the forensic discrimination.

The emission spectra of luminescent trivalent europium (Eu3+) and terbium (Tb3+) complexes were measured using a microscope laser Raman spectrometer with a doubled Nd:YAG laser (532 nm) and an Ar laser (488 nm). Excitation at 532 and 488 nm corresponded to wavelengths of the 7F1 --> 5D1 band of Eu3+ and the 7F6 --> 5D4 band of Tb3+, respectively. The Eu3+ and Tb3+ complexes were discriminated by high-resolution emission spectra more distinctly and sensitively than by fluorescence spectrometry, the usual analytical method.     

ANALYSIS OF LASER-INDUCED FLUORESCENCE LINE SHAPE OF INTACT LEAVES: APPLICATION TO UV STRESS DETECTION

Abstract— In vivo laser-induced fluorescence spectra of intact leaves of healthy and UV-irradiated Salvia splendens plants excited at 337 nm by a nitrogen laser were recorded using an optical multichannel analyzer system. The spectra showed the typical fluorescence bands centered around 450, 530, 685 and 730 nm. Exposure to UV radiation changed the relative intensity values of these bands and their peak positions. The analysis of the acquired spectra in terms of a linear combination of Gaussian bands was carried out to determine accurately the peak positions and the relative intensity contribution of the various bands to the laser-induced fluorescence spectra on healthy and UV-treated plants of different age.
The results indicate that a curve-fitting analysis of the measured fluorescence spectra is a useful and sensitive method to discriminate the various band contribution to the whole leaf fluorescence spectrum. The comparison among blue-green and red-far-red fluorescence of leaves was also confirmed as an effective indicator of UV stress in plants.     

FLUORESCENCE QUENCHING OF ALLOPHYCOCYANIN AND PHYTOCHROME BY ESTRIOL*

Abstract— At 293 K the long-wavelength absorption and emission band of 1.4 μM allophycocyanin is decreased by estriol (Δ¹-³-⁵⁽¹⁰⁾-estratriene-3,16α, 17β-trio!) in the range 0.8-6.6 μM in the presence of 11% alcohol (vol/vol). The binding of estriol is shown to be of high affinity, 1:1 with allophycocyanin. The free energy of this binding process (ΔG^°) is -33.6 kJ mol' and single binding site dissociation constant (K_D) 1.0 ×10–6M. Estriol at 21 μM effectively quenches the fluorescence of 1.4 M large molecular weight phytochrome in its red absorbing form at 77 K while having little or no effect on the phototransformation difference spectrum at 293 K.     

Simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef-building corals

Light is absorbed by photosynthetic algal symbionts (i.e. zooxanthellae) and by chromophoric fluorescent proteins (FP) in reef‐building coral tissue. We used a streak‐camera spectrograph equipped with a pulsed, blue laser diode (50 ps, 405 nm) to simultaneously resolve the fluorescence spectra and kinetics for both the FP and the zooxanthellae. Shallow water (<9 m)–dwelling Acropora spp. and Plesiastrea versipora specimens were collected from Okinawa, Japan, and Sydney, Australia, respectively. The main FP emitted light in the blue, blue‐green and green emission regions with each species exhibiting distinct color morphs and spectra. All corals showed rapidly decaying species and reciprocal rises in greener emission components indicating Förster resonance energy transfer (FRET) between FP populations. The energy transfer modes were around 250 ps, and the main decay modes of the acceptor FP were typically 1900–2800 ps. All zooxanthellae emitted similar spectra and kinetics with peak emission (～683 nm) mainly from photosystem II (PSII) chlorophyll (chl) a. Compared with the FP, the PSII emission exhibited similar rise times but much faster decay times, typically around 640–760 ps. The fluorescence kinetics and excitation versus emission mapping indicated that the FP emission played only a minor role, if any, in chl excitation. We thus suggest the FP could only indirectly act to absorb, screen and scatter light to protect PSII and underlying and surrounding animal tissue from excess visible and UV light. We conclude that our time‐resolved spectral analysis and simulation revealed new FP emission components that would not be easily resolved at steady state because of their relatively rapid decays due to efficient FRET. We believe the methods show promise for future studies of coral bleaching and for potentially identifying FP species for use as genetic markers and FRET partners, like the related green FP from Aequorea spp.