Studies on the Localization and Spectral Characteristics of the Fluorescence Emission of Differently Pigmented Wheat Leaves

Intensity, spectral characteristics and localization of the UV-laser (337 nm) induced blue-green and red fluorescence emission of green, etiolated and white primary leaves of wheat seedlings were studied in a combined fluorospectral and fluoromicroscopic investigation. The blue-green fluorescence of the green leaf was characterized by a maximum near 450 nm (blue region) and a shoulder near 530 nm (green region), whereas the red chlorophyll fluorescence exhibited maxima in the near-red (F690) and far-red (F735). The etiolated leaf with some carotenoids and traces of chlorophyll a, in turn, showed a higher intensity of the blue-green fluorescence with a shoulder in the green region and a strong red fluorescence peak near 684 to 690 nm, the far-red chlorophyll fluorescence maximum (F735) was, however, absent. The norfluorazone-treated white leaf, free of chlorophylls and carotenoids, only exhibited blue-green fluorescence of a very high intensity. In green and etiolated leaves the blue-green fluorescence primarily derived from the cell walls of the epidermis and the red fluorescence from the chlorophyll a of the mesophyll cells. In white leaves the blue-green fluorescence emanated from all cell walls of epidermis, mesophyll and leaf vein bundles. The shape and intensity of the blue-green and red fluorescence emission is determined by the reabsorption properties of chlorophylls and carotenoids in the mesophyll, thus giving rise to quite different values of the various fluorescence ratios F450/F690, F450/F530, F450/F735 and F690/F735 in green and etiolated leaves.     

Characterization of the laser-induced blue, green and red fluorescence signatures of leaves of wheat and soybean grown under different irradiance

The blue, green and red fluorescence emission of green wheat ( Triticum aestivum L. var. Rector) and soybean leaves ( Glycine max L. var. Maple Arrow) as induced by UV light (nitrogen laser: 337 nm) was determined in a phytochamber and in plants grown in the field. The fluorescence emission spectra show a blue maximum near 450 nm, a green shoulder near 530 nm and the two red chlorophyll fluorescence maxima near 690 and 735 nm. The ratio of blue to red fluorescence, F450/F690, exhibited a clear correlation to the irradiance applied during the growth of the plants. In contrast, the chlorophyll fluorescence ratio, F690/F735, and the ratio of blue to green fluorescence, F450/F530, seem not to be or are only slightly influenced by the irradiance applied during plant growth. The blue fluorescence F450 only slightly decreased, whereas the red chlorophyll fluorescence decreased with increasing irradiance applied during growth of the plants. This, in turn, resulted in greatly increased values of the ratio, F450/F690, from 0.5 – 1.5 to 6.4 – 8.0. The decrease in the chlorophyll fluorescence with increasing irradiance seems to be caused by the accumulation of UV light absorbing substances in the epidermal layer which considerably reduces the UV laser light which passes through the epidermis and excites the chlorophyll fluorescence of the chloroplasts in the subepidermal mesophyll cells.     

Effects of light quality on chlorophyll-forms Ca 684, Ca 690 and Ca 699 of the diatom Phaeodactylum tricornutum

Colored light modifies the relative concentration of chlorophyll-forms of the diatom Phaeodactylum tricornutum compared to white-light control. No change in the ratio carotenoids/chlorophylls was observed after 4 days exposure to green light (max: 530 nm), blue light (max: 470 nm) or red light ( > 650 nm) of same intensity.However, the absorption spectra were modified, the content in Ca 684, Ca 690, Ca 699 forms increased in red and green light cultures and photosynthetic unit size of PS II decreased by 30% in green and blue light cultures.Fluorescence emission and fluorescence excitation spectra according to the Butler and Kitajima method (1975) were carried out for each culture. Ca 669 form was predominant in the two photosystems. The newly appeared far red forms fluoresce at 715 nm like PS I forms.We conclude that these new forms originated in a rearrangement of PS II forms. They do not transmit excitation energy to reaction center of PS I and are disconnected from the other chlorophyll-forms of the photosynthetic antennae.Abbreviations ABS absorption - Ca chlorophyll-complex - chla chlorophyll a - chl c chlorophyll c - chl t total chlorophylls - D.C.M.U. 3-(3, 4 dichlorophenyl) 1-diméthyl-urea - d^v division - F fluorescence - PS I and PS II photosystem I and photosystem II     

Subcellular detection of lipase activity in plant protoplasts using fluorescence microscopy

Measurement of lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) activity in tissue extracts often poses problems due to the inhibitors released during homogenization. The present investigation provides a first report on the localization of lipase activity in viable plant protoplasts isolated from the cotyledons of germinating seeds of sunflower, cotton and peanut by fluorescence microscopy, using a lipase specific, 'glycerol-derived' synthetic substrate, i.e., 1,2-O-dilauryl-rac-3-glycero-glutaric acid-resorufin ester, commonly used for in vitro assays. Due to its lipophilicity, this chromogenic substrate readily permeates plasma membrane of plant protoplasts. Subsequent lipase action leads to cleavage of this substrate to release resorufin, which can be visualized due to emission of red fluorescence (^max excitation – 567 nm; ^max emission – 584.6 nm) at its intracellular locations.     

Investigations of the Blue-green Fluorescence Emission of Plant Leaves

The UV light (337 nm) induced blue-green fluorescence emission of green leaves is characterized at room temperature (298 K) by a maximum near 450 nm (blue region) and a shoulder near 525 nm (green region) and was here also studied at 77 K. At liquid nitrogen temperature (77 K) the blue (F450) and green fluorescence (F525) are much enhanced as is the red chlorophyll fluorescence near 735 nm. During development of green tobacco leaves the blue fluorescence F450 (77 K) is shifted towards longer wavelengths from about 410 nm to 450 nm. The isolated leaf epidermis of tobacco showed only slight fluorescence emission with a maximum near 410 nm. The green fluorescence F525 was found to mainly originate from the mesophyll of the leaf, its intensity increased when the epidermis was removed. The red chlorophyll fluorescence emission was also enhanced when the epidermis was stripped off; this considerably changed the blue/red fluorescence ratios F450/F690 and F450/F735. The epidermis, with its cell wall and UV-light-absorbing substances in its vacuole, plays the role of a barrier for the exciting UV-light. In contrast to intact and homogenized leaves, isolated intact chloroplasts and thylakoid membranes did not exhibit a blue-green fluorescence emission.     

Studies on the constancy of the blue and green fluorescence yield during the chlorophyll fluorescence induction kinetics (Kautsky effect)

Blue (F 450) and green (F 530) leaf fluorescence were studied together with the red chlorophyll fluorescence (emission maxima F 690 and F 735) during light-induced chlorophyll fluorescence induction kinetics (Kautsky effect) in predarkened leaves of wheat (Triticum aestivum L.) and soybean (Glycine max L.). The intensity of the red chlorophyll fluorescence decreased from maximum fluorescence Fm to steady-state fluorescence Fs, and the fluorescence ratio F 690/F 735 decreased by about 10% from Fm to Fs. However, blue and green fluorescence intensities remained constant throughout the measuring time. Consequently, the ratio of blue to red fluorescence (F 450/F 690) increased during chlorophyll fluorescence induction kinetics, whereas the ratio of blue to green fluorescence (F 450/F 530) remained unchanged within the same period. The knowledge of these ratios will be a prerequisite for the interpretation of remote sensing data from terrestrial vegetation.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

Multicolour Fluorescence Imaging of Sugar Beet Leaves with Different Nitrogen Status by Flash Lamp UV-Excitation

Fluorescence images of leaves of sugar beet plants (Beta vulgaris L. cv. Patricia) grown on an experimental field with different fertilisation doses of nitrogen [0, 3, 6, 9, 12, 15 g(N) m^–2] were taken, applying a new multicolour flash-lamp fluorescence imaging system (FL-FIS). Fluorescence was excited by the UV-range (280–400 nm, _max = 340 nm) of a pulsed Xenon lamp. The images were acquired successively in the four fluorescence bands of leaves near 440, 520, 690, and 740 nm (F₄₄₀, F₅₂₀, F₆₉₀, F₇₄₀) by means of a CCD-camera. Parallel measurements were performed to characterise the physiological state of the leaves (nitrogen content, invert-sugars, chlorophylls and carotenoids as well as chlorophyll fluorescence induction kinetics and beet yield). The fluorescence images indicated a differential local patchiness across the leaf blade for the four fluorescence bands. The blue (F₄₄₀) and green fluorescence (F₅₂₀) were high in the leaf veins, whereas the red (F₆₉₀) and far-red (F₇₄₀) chlorophyll (Chl) fluorescences were more pronounced in the intercostal leaf areas. Sugar beet plants with high N supply could be distinguished from beet plants with low N supply by lower values of F₄₄₀/F₆₉₀ and F₄₄₀/F₇₄₀. Both the blue-green fluorescence and the Chl fluorescence rose at a higher N application. This increase was more pronounced for the Chl fluorescence than for the blue-green one. The results demonstrate that fluorescence ratio imaging of leaves can be applied for a non-destructive monitoring of differences in nitrogen supply. The FL-FIS is a valuable diagnostic tool for screening site-specific differences in N-availability which is required for precision farming.     

Imaging of the Blue,Green, and Red Fluorescence Emission of Plants: An Overview

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.     

Regulation of the distribution of excitation energy in Ochromonas danica,an organism containing a chlorophyll-A/C/carotenoid light harvesting antenna

A chlorophyll a, c-fucoxanthin pigment-protein complex8 functions as the major light harvesting antenna in the Chrysophyte Ochromonas danica. The regulated distribution of excitation energy between the two photosystems was investigated in these organisms and was shown to be strongly wavelength dependent. A light state transition was induced by pre-illumination of cells using light 2 (640 nm) and light 1 (700 nm) of equal absorbed intensity, and detected by reversible changes in the 77 K chlorophyll fluorescence emission spectra. Peaks at 690 nm and 720 nm in the low temperature spectra are most likely associated with PS2 and PS1 respectively. A room temperature fluorescence emission at 680 nm induced by modulated light 2 (500 nm) was strongly quenched in the presence of background light 1 (720 nm). Removal of light 1 led to an increase in fluorescence followed by a slow quenching. The room temperature fluorescence changes were directly correlated with changes in the 77 K emission spectra that indicated a change in the distribution of excitation energy between the two photosystems. It was established that DCMU (1 mol) prevented the state 2. The conversion to state 1 followed a simple photochemical dose dependence and had a half-time of 20 s-1.5 min at 6 W m^-2. In contrast, the conversion to state 2 was independent of light intensity. These data indicate that O. danica undergoes a light state transition in response to the preferential excitation of PS2 or PS1.Abbreviations PS2 photosystem 2 - PS1 photosystem 1 - LHC light harvesting chlorophyll a/b protein - fx fucoxanthin - PQ plastoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea     

In vivo fluorometric method for early detection of cyanobacterial waterblooms

A specific method was developed for monitoring the concentration of cyanobacteria (blue-green algae) before waterblooms, based on their characteristics ofin vivo fluorescence. The excitation and emission spectra of cyanobacteria are very different from those of eukaryotic algae, due to the importance of phycocyanin, rather than chlorophylla, in determining the fluorescence characteristics. Our results, based on four cyanobacteria:Microcystis aeruginosa, Anabaena cylindrica, Phormidium tenue andSpirulina platensis, indicate that excitation at 620 nm and its emission at 645 nm is a sensitive and specific method for their detection. Furthermore, the addition of 10 M photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) induced only 3% increase in phycocyanin fluorescence, suggesting that this measurement is almost independent of the ongoing rate of photosynthesis.Author for correspondence     

Photopigments of the lateral eye ofLimulus

Summary Photoreceptor membrane fractions of the lateral eye ofLimulus were solubilized in the detergent emulphogene, and three photobleachable materials were observed with respective _max values at 330nm±10nm, 450 nm±10 nm, and 530 nm±10 nm. A530 is the pigment which had been reported earlier by Hubbard and Wald (1960), and it can be separated from A330 and A450 on the basis of differential solubility in digitonin. Approximately the same number of incident quanta were required for a unit absorbance change at _max for all three pigments, but measurable photo-products were not observed after bleaching A330 and A450.We thank Daniel Inners for discussion and Thomas Wheeler and Vivian Leitner for assistance. This work was supported by NSF grants GB-33499 and BMS 75-07197, NIH grants EY-00871 and EY-00244, and The Institute of Ophthalmology, Houston. We also thank I.L. and Bertha Gordon Miller for their generous gift of the Cary 118C.     

On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (_max 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (_max 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.Abbreviations GCP guard-cell protoplast - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Some improvements in calculating the plant stand surface albedo and its influence on ground surface temperature

It is shown that better results are obtained in calculating the surface albedo of a plant stand for near-infrared solar radiation using the modified two-stream approximation in favor of the Schwarzschild one. These evaluations of the plant stand surface albedo were obtained by assuming the values for scattering parameters of upward ₀ and diffusive solar radiation in the plant stand, by applying the delta-Eddington approximation. However, more accurate evaluations of ₀ and can be obtained by using Rayleigh's phase scattering functions for radiative scattering in a plant stand. Furthermore, much attention is devoted to testing the influence of the plant stand albedo for the visible part of solar radiation (for which the Schwarzchild approximation is valid) on ground surface temperature. The ground surface temperature was calculated by the force-restore method; the greatest ground surface temperature corrections were obtained in cases where the surface beneath the plant stand is covered with snow. The influence on ground surface temperature of high absorption and scattering in dense plant stands with horizontal foliage was also tested. Again significant corrections of ground surface temperature were obtained, taking into consideration the given characteristics of a plant stand compared to a standard albedo value.List of symbols A albedo of direct radiation in plant canopy - A _D albedo of diffuse radiation in plant canopy - A _p albedo of the ground surface - A _Q total albedo in plant canopy - a proportion of gaps in plant canopy characteristic of surface evaporation - _w constant - parameter of upward scattering diffuse radiation - ₀ parameter of upward scattering direct radiation - C _g soil volumetric heat capacity - C _p air specific heat - C _DH aerodynamic transfer coefficient for heat - C _DE aerodynamic transfer coefficient for moisture - d depth of soil - sun's declination - azimuth of incident radiation - azimuth of diffuse radiation - characteristic of the radiative sources - G foliage orientation function - g parameter in delta-Eddington approximation - g _z parameter for near-infrared radiation - _L scattering phase function radiation - _D diffuse solar radiation flux - _G soil heat flux - _H air sensible heat flux - _L air latent heat flux - _O solar constant - _R net longwave radiation flux - _S direct solar radiation flux - h _o height of the sun - i intensity of diffuse radiation - k von Karman constant - L leaf area index - L _v coefficient for latent heat of evaporation - _g soil heat conductivity - n/N relative cloudiness - solid angle - frequency of temperature wave - Q _a specific humidity of air - Q _w specific humidity of saturated air - R _L coefficient of plant reflectivity - r direction of diffuse radiation - direction of incident radiation - r _o direction of solar radiation - r _s resistance of the leaf stomata - p _o air density - S intensity of direct solar radiation - Stefan-Boltzman constant - T _a air temperature - T _G ground surface temperature - T _c cloud temperature - T ₁ feaf temperature - T _* air temperature scale - t time - t time step - zenith angle of diffuse radiation - zenith angle of incident radiation - ₀ zenith angle of solar radiation - U _* friction velocity scale - V near-surface wind velocity - W _g soil water content - W _L scattering coefficient in plant canopy - W _sat saturated soil water content - z vertical direction     

Presence of a class of chromophores as monitor of oxygen-linked conformational changes in hemocyanins

Summary Oxygenation of native hemocyanins fromHelix pomatia andPanulirus interruptus under conditions of cooperative binding, causes a change in the dynamic behaviour of the internal structure, leading to increased rotational mobility of a class of tryptophan residues emitting above 450 nm. This is associated with the complete depolarization of the emission on a time scale where the large hemocyanin is practically immobile. This class is thought to be very near the active site since it is strongly affected by the copper atoms. Moreover, fluorescence changes of the class of chromophores emitting above 450 nm is more marked in the molluscanHelix hemocyanin than in the arthropodanPanulirus hemocyanin, suggesting a possible difference in the structure of the active site or in the extent of the allosteric transition between the two species. This class of chromophores may by useful probes to monitor ligand-linked conformational change in hemocyanins.     

Biochemical and spectroscopic analysis of UV effects in the marine flagellate Cryptomonas maculata

The effects of solar and artifical ultraviolet radiation on the marine cryptoflagellate, Cryptomonas maculata, were studied. Even after short exposure to UV the accessory photosynthetic pigment phycoerythrin is bleached; likewise the fluorescence undergoes significant changes both in amplitude and in the maximal peak wavelength. In parallel, the photosynthetic oxygen production decreases rapidly during exposure. Gel electrophoresis and FPLC of membrane proteins show a significant decrease in chromoproteins after 2 h UV, which is confirmed by fluorescence excitation and emission spectra of the FPLC fractions.Abbreviations APS ammonium persulfate - DCMU 3-(3,4dichlorophenyl)1,1-dimethylurea; Emulphogen, polyoxyethylene 10 tridecyl ether - FPLC fast protein liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - SDS PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - TEMED NN NNtetramethylethylene diamine - UV-A wavelength range between 320 nm and 400 nm - UV-B wavelength range between 280 nm and 320 nm Dedicated to the 60th birthday of Professor Dr. W. Wehrmeyer     

Remote Sensing of Solar-excited Plant Fluorescence as a Measure of Photosynthetic Rate

Leaf level net photosynthetic rates (P _N) of laurel oak (Quercus hemispherica) juveniles grown under contrasting nutrient and CO₂ regimes were negatively correlated with red to far-red ratios, R/FR (690/760 nm), steady-state, solar-excited fluorescence ratios (r ² = 0.66, n = 12) measured across 12 plant canopies. Laurel oak juveniles that had been subjected to nitrogen stress over a period of a year demonstrated higher R/FR than their counterparts that had been provided with sufficient nitrogen. Plants that had been grown at elevated CO₂ concentrations, EC [700 mol (CO₂) mol^-1] also exhibited significantly higher R/FR when subjected to normal ambient carbon dioxide concentrations than their counterparts grown under ambient concentrations, AC [380 mol (CO₂) mol^-1]. All fluorescence measurements were obtained by observing a multi-plant canopy using a unique solar-blind passive sensor. This sensor, which utilizes Fraunhofer-line discrimination techniques, detects radiation at the cores of the lines comprising the atmospheric oxygen A- and B-bands, centered at 762 and 688 nm, respectively. These results support the use of solar-excited steady-state plant fluorescence as a potential tool for remote measurement of canopy radiation use efficiency.     

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy

Summary MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450–500 nm excitation/>510 nm emission) or rhodamine (514 nm excitation/>530 emission) type dyes. A second 5- to 10-fold brighter emission for 450–500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled domes and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 m riboflavin resulted in accumulation in domes of 565±80 m. The transport rate was calculated to be 189±30 pmol/min-cm². Onemm probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10mm nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatable with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.     

Fluorescence detected magnetic resonance of the primary donor and inner core antenna chlorophyll in Photosystem I reaction centre protein: Sign inversion and energy transfer

The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state ³P700, created via radical pair formation and recombination. ³P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at _f > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10^-4 cm^-1 and E=38.5×10^-4 cm^-1, were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the ³P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < _f < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < _f < 700 nm (inner core antennae, F690), and _f > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of ³P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.Abbreviations Chl chlorophyll - CP1 core chlorophyll protein of Photosystem I - EPR electron paramagnetic resonance - F690, F720 chlorophyll forms having fluorescence maximum at 690–695 and 720 nm, respectively - F(A)(O)DMR fluorescence (absorption) (optical) detected magnetic resonance - FF fluorescence fading - ISC intramolecular intersystem crossing - _f fluorescence emission wave-length - LHC I light harvesting chlorophyll a/b protein of Photosystem I - P700 primary donor of Photosystem I - PS I Photosystem I - RC reaction centre - RP radical pair - SDS sodium dodecyl sulphate - ZFS zero field splitting     

Blue,Green and Red Fluorescence Signatures and Images of Tobacco Leaves*

Laser-induced fluorescence images of the leaf of an aurea mutant of Nicotiana tabacum were recorded for the blue and green fluorescence at 440 and 520 nm and the red chlorophyll fluorescence at 690 and 735 nm. The results obtained were compared with direct measurements of the fluorescence emission spectra of leaves using a conventional spectrofluorometer. The highest emission of blue (F440) and green fluorescence (F520) within the leaf was found in the leaf veins, particularly the main leaf vein. In contrast, the intercostal fields of leaves, which exhibited the highest chlorophyll content, showed only a very low blue and green fluorescence emission, which was much lower than the red and far-red chlorophyll fluorescence emission bands (F690 and F735). Correspondingly, the ratio of blue to red leaf fluorescence F440/F690 of upper and lower leaf side was much higher in the leaf veins (values 1.2 to 1.5) than in intercostal fields (values of 0.6 to 0.7). The results also demonstrated that in the intercostal fields the major part of the blue-green fluorescence was reabsorbed by chlorophylls and carotenoids. A partial reabsorption of the red fluorescence band near 690 nm by leaf chlorophyll took place, but did not affect the far-red fluorescence band near F735. As a consequence the chlorophyll fluorescence ratio F690/F735 exhibited significantly higher values in the chlorophyll-poor leaf vein regions (1.7 to 1.8) than in the chlorophyll-rich intercostal fields (0.8 to 1.3). Imaging spectroscopy of leaves was shown to be much more precise than the screening of fluorescence signatures by conventional fluorometers. It clearly demonstrated that the blue-green fluorescence and the red chlorophyll fluorescence of leaves exhibit an inverse contrast to each other. The advantage of the fluorescence imaging spectroscopy, which allows the simultaneous screening of the whole leaf surface and distinct parts of it, and its possible application in the detection of stress effects or local damage by insects and pathogens, is discussed.