Fluorescence of neurotransmitters and their reception in plant cell

The use of fluorescent reagents for the histochemical detection of catecholamines or histamine, as well as luminescent antagonists of the intracellular neurotransmitters revealed that they can bind to certain cellular compartments. After the treatment with glyoxylic acid (a reagent used for the detection of catecholamines), blue fluorescence with maximum at 460–475 nm was visualized in nuclei and chloroplasts (in control preparations no emission in this spectral region was recorded), as well as an intense fluorescence, exceeding the control level, in the vacuoles. After the exposure to ortho-phthalic aldehyde (a reagent used for the histamine detection), blue emission was more noticeable in nuclei and chloroplasts, which correlates with previously observed effects on intact cells, such as pollen and vegetative microspores. A comparison of the intensities of the biogenic amine-related emission in various organelles showed that the greatest emission was in vacuoles and the weakest, in chloroplasts. Thus, on the surface, and possibly within the organelles, fluorescence could demonstrate the presence of biogenic amines. Antagonists of the neurotransmitters (dtubocurarine for acetylcholine; yohimbine for dopamine; norepinephrine and inmecarb for serotonin), which fluoresce in the blue and blue-green region and usually bind with the plasmalemma of intact cells, also interacted with the membranes of the organelles studied. Fluorescence intensity depended on the object; most prominent it was for yohimbine in the outer membrane of the nucleus, vacuoles, and chloroplasts.     

Chemical signaling in plant microspore cells

Chemical signal transduction from the cell surface to organelles was studied in unicellular vegetative (Equisetum arvense) and generative (Hippeastrum hybridum pollen) microspores of plants. Neurotransmitters acetylcholine, dopamine, and serotonin, their agonists and antagonists, Na⁺, K⁺, and Ca²⁺ channel blockers, as well as forskolin and theophylline (agents increasing the intracellular level of cyclic adenosine monophosphate) were used as chemical signals. Both types of microspores exposed to neurotransmitters, their agonists, forskolin, and theophylline demonstrated growth activation, while neurotransmitter antagonists and ion channel blockers inhibited this process. No stimulating effects of neurotransmitters were observed for cells pretreated with the antagonists and ion channel blockers. Pretreatment with ion channel blockers and then by anticontractile agents (cytochalasin B or colchicine) either had no effect or increased the inhibition of microspore growth. Pathways of chemical signal transduction from the cell surface to organelles are discussed.     

Contractile Proteins in Chemical Signal Transduction in Plant Microspores

Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using unicellular systems. Neurotransmitters dopamine and serotonin as well as active oxygen species hydrogen peroxide and tert-butyl peroxide were used as chemical signals. Experiments were carried out on vegetative microspores of field horsetail Equisetum arvense and generative microspores (pollen) of knight’s star Hippeastrum hybridum treated with cytochalasin B (an inhibitor of actin polymerization in microfilaments), colchicine, and vinblastine (inhibitors of tubulin polymerization in microtubules). Both types of the treated microspores demonstrated suppressed development, particularly, after cytochalasin B treatment. At the same time, increased blue fluorescence was observed in certain cell regions (along the cell wall and around nuclei and chloroplasts) where the corresponding contractile proteins could be localized. In contrast to anticontractile agents, dopamine, serotonin B, and peroxides stimulated microspore germination. Microspore pretreatment with cytochalasin B and colchicine followed by the treatment with serotonin, dopamine, or peroxides decreased the germination rate. The involvement of actin and tubulin in chemical signal transduction from the cell surface to the nucleus is proposed.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 281–286.Original Russian Text Copyright © 2005 by Roshchina.     

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.     

Autofluorescence of intact Equisetum arvense L. spores during their development

The autofluorescence of horsetail Equisetum arvense spores excited with UV-light of 360-380 nm was studied by microspectrofluorimetry during their development from an individual cell to the formation of a multicellular thallus with the generative organs. The investigation involved the registration of the fluorescence spectra of individual intact developing cells and the measurement of the ratio of cell fluorescence intensities in the blue and red regions of the spectrum. Dry blue-fluorescing microspores showed the maxima at 460 and 530 nm and a small maximum at 680 nm. Thirty minutes after moistening in water, red-fluorescing cells arose among blue-fluorescing microspores, indicating the onset of development. Red fluorescence with a maximum at 680 nm enhanced as cells put off their cover, which brightly fluoresced in the blue region of the spectrum with the main maximum at 460 nm. By estimating the ratio of autofluorescence intensities in the blue region of the spectrum to red lightening of microspores at the first stages of development up to 24 h (in particular, their first division, the formation of nonfluorescencing rhizoid, etc.), nonviable (only blue-lightening) cells were distinguished from viable cells, in which red fluorescence began to prevail. After 25-40 days of development, the gametophyte fluorescing mainly at 680 nm formed male organs, antheridia, with blue-green-fluorescing spermatozoids. Then female generative organs archegonia with the egg cell appeared, which fluoresced blue, whereas the surrounding cells fluoresced red. It was supposed that the lightening in the blue and green regions of the spectrum is due to the presence of phenols, terpenoids, and azulenes, whereas the emission in the red region is associated with the presence of chlorophyll and azulenes. The observation of autofluorescence makes it possible to easily distinguish generative cells without additional staining.     

Identification of acetylcholine and impact of cholinomimetic drugs on cell differentiation and growth in the unicellular green alga Micrasterias denticulata

Acetylcholine (ACh), one of the best-studied neurotransmitters, has been reported in animals as well as in multicellular plants. In higher plants, ACh affects phytochrome-dependent growth and differentiation. In the present study on the green alga Micrasterias denticulata which has been used as a cell biological model system since several decades, we identified ACh for the first time in a unicellular alga, demonstrated light as a regulatory factor of ACh production and showed that cholinergic agonists and antagonists suppress growth and differentiation. ACh was detected in Micrasterias cells grown under light–dark conditions of 14/10 h, but not in dark-grown algae, by high-performance liquid chromatography coupled with mass spectrometry. To quantify cholinergic effects on cell differentiation, we exposed young developmental stages of Micrasterias to cholinergic antagonists (d-tubocurarine, hexamethonium) as well as agonists (carbachol, nicotine). We found that the cholinergic antagonists and, surprisingly, the agonist nicotine significantly suppressed cell growth and differentiation in a dose-dependent manner. Moreover, we demonstrated that secondary wall formation is specifically inhibited in the presence of nicotine.     

Growth control by phytochrome of de-etiolated bean hypocotyls mediated by chlorophyll fluorescence

The elongation of hypocotyls excised from de-etiolated seedlings of beans (Phaseolus vulgaris L. cv. British Wax) is inhibited by light, blue and red irradiations being equally effective. Conditions which decrease chlorophyll fluorescence, such as CO₂-free air, abolish the inhibitory effect of blue irradiation and enhance the inhibition by red light. Conversely, conditions which increase chlorophyll fluorescence, such as a N₂ atmosphere or irradiation through a chlorophyll filter, abolish the inhibitory effect of red light and enhance the inhibition by blue irradiation. The inhibitory effect of blue light is reversible by red irradiation under increased fluorescence as well as by far red. We propose that the chlorophyll fluorescence excited by blue and red irradiations in λ_F > 660 nm and λ_F > 720 nm, respectively, is responsible for the inhibitory effect of blue light and the reduction of the inhibitory effect of non fluorescing red light. Both red and blue wavelengths seem, therefore, to control hypocotyl elongation through phytochrome.     

Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system

A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF, we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, R_Fd (defined as Chl fluorescence decrease F_d from maximum to steady state fluorescence F_s:F_d/F_s), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio R_Fd is linearly correlated to the net photosynthetic CO₂ fixation rate P _N as is indicated here for sun and shade leaves of various trees that considerably differ in their P _N. Imaging the R_Fd-ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F₆₉₀. Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F₄₄₀/F₆₉₀, whereas differences in Chl content are detected by collecting images of the fluorescence ratio red/far-red, F₆₉₀/F₇₄₀.     

Effects of different excitation and detection spectral regions on room temperature chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence parameters

The effects of different spectral region of excitation and detection of chlorophyll (Chl) a fluorescence at room temperature on the estimation of excitation energy utilization within photosystem (PS) 2 were studied in wild-type barley (Hordeum vulgare L. cv. Bonus) and its Chl b-less mutant chlorina f2 grown under low and high irradiances [100 and 1 000 μmol(photon) m⁻² s⁻¹]. Three measuring spectral regimes were applied using a PAM 101 fluorometer: (1) excitation in the red region (maximum at the wavelength of 649 nm) and detection in the far-red region beyond 710 nm, (2) excitation in the blue region (maximum at the wavelength of 461 nm) and detection beyond 710 nm, and (3) excitation in the blue region and detection in the red region (660– 710 nm). Non-photochemical quenching of maximal (NPQ) and minimal fluorescence (SV₀), determined by detecting Chl a fluorescence beyond 710 nm, were significantly higher for blue excitation as compared to red excitation. We suggest that this results from higher non-radiative dissipation of absorbed excitation energy within light-harvesting complexes of PS2 (LHC2) due to preferential excitation of LHC2 by blue radiation and from the lower contribution of PS1 emission to the detected fluorescence in the case of blue excitation. Detection of Chl a fluorescence originating preferentially from PS2 (i.e. in the range of 660–710 nm) led to pronounced increase of NPQ, SV₀, and the PS2 photochemical efficiencies (F_V/F_M and F_V′/F_M′), indicating considerable underestimation of these parameters using the standard set-up of PAM 101. Hence PS1 contribution to the minimal fluorescence level in the irradiance-adapted state may reach up to about 80 %.     

Laser-induced fluorescence (LIF) spectra of herbaceous and woody pre- and post-digested plant material

Filtrate from pre- and post-digested plant material was exposed to 355-nm pulsed laser light and the subsequent laser-induced fluorescence (LIF) was recorded. Similarities and differences among spectra from 20 materials are discussed. Each material was replicated once, dried, ground, and exposed to chloroform (CHCl₃) for 24 h. The material represented aged (1 to 18 years old) plants from different herbaceous (grasses and forbs) and woody plant life forms. Mean peak fluorescence recorded among materials differed (P<0.0001) in both wavelength and peak amplitude (counts) across the spectral range (387 to 788 nm). Peak fluorescence was evaluated within each of three arbitrary color categories, blue near 455 nm and red near 674 nm, while only 16 of the materials produced a green peak near 528 nm. In general, the blue and green fluorescence peaks were broad while the red peak was narrow. Mean peak counts were largest in the red range. Varying amounts of laser beam absorption occurred among the materials evaluated due to different concentrations of filtrate and different absorption efficiencies; therefore, amplitude data (counts) were not used to determine statistical differences among materials. To overcome difficulties attributed to the raw count data, red/blue, red/green and blue/green count ratios within replicates were calculated. Using all three count ratios in a multivariate analysis of variance, the 16 materials could be separated into nine different (P<0.05) material groupings. The LIF technique may provide a reliable means to separate ground pre- and post-digested plant materials following further research into determining what fluorophores are producing the spectral signatures and how sample preparation affect peak wavelengths.     

Evaluating UV-B effects and EDU protection in cucumber leaves using fluorescence images and fluorescence emission spectra

A newly developed laboratory fluorescence imaging system was used to obtain fluorescence images (FImage) of freshly excised cucumber (Cucumis sativus L.) leaves in spectral bands centered in the blue (F450), green (F550), red (F680), and far-red (F730) spectral regions that resulted from a broad-band (300-400 nm) excitation source centered at 360 nm. Means of relative fluorescence intensities (RFI) from these spectral fluorescence images were compared with spectral fluorescence emission data obtained from excitation wavelengths at 280 nm (280EX, 300-550 nm) and 380 nm (380EX, 400-800 nm) of dimethyl sulfoxide (DMSO) extracts from these leaves. All three fluorescence data types (FImage, 280EX, 380EX) were used to assess ultraviolet-B (UV-B, 280-320 nm) induced physiological changes and the possible use of N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N′-phenylurea (EDU or ethylenediurea) as a chemical protectant against UV-B damage. Plants exhibited well known foliar growth and pigment responses to UV-B exposure (e.g., increased UV-B absorbing compounds and decreased leaf area, chlorophyll a content; and and lower chlorophyll a/b and chlorophyll/carotenoid pigment ratios). Since EDU alone had no effect on foliar variables, there was no evidence that EDU afforded protection against UV-B. Instead, EDU augmented some UV-B effects when provided in conjunction with UV-B irradiation (e.g., reductions in the chlorophyll/carotenoid ratio, total photosynthetic pigments, and chlorophyll b content).Relative fluorescence intensities (RFI) in the longer visible wavelengths (green, red, and far-red) were uncorrelated for comparisons between the FImage and 380EX data sets. However, blue and green RFI were significantly correlated (0.8r0.6; P ≤0.002) for comparisons between FImage and 280EX data sets. UV-B treatment caused an increase in blue RFI (e.g., F450) in both images and 280EX measurements. One explanation is that the UV-B excitation of both 280EX and FImage stimulates processes that produce excess blue fluorescence. The molecules that produce the excess blue fluorescence in both the 280EX and the Fimage data are different electron transfer agents that operate in parallel. For FImage, the UV excitation penetrates leaf surface layers to stimulate fluorescence from compounds in mesophyll and epidermal tissues (as occurs for the extracts of leaf discs), whereas emissions captured at longer, less energetic wavelengths, were primarily from the epidermal layer. UV-B irradiated leaves showed much greater heteorgeneity of RFI in both the green (F550_FImag) and the red (F680_FImag) bands than unirradiated leaves; this was true irrespective of EDU treatment.Although qualitative responses in individual bands differed between FImage and 380EX data, similar results were obtained in the detection of UV-B induced effects when the red/green and blue/far-red fluorescence ratios of these data were compared. The red/green ratio (either F680/F550_FImage or F675/F525_380EX) was lower for UV-B exposed plants in both images and 380EX data. UV-B exposure also significantly enhanced the blue/far-red ratio of images (F450/F740_FImage) and the comparable 380EX ratio (F450/F730_380EX) for the combined UV-B/EDU group. The far-red/red ratios were not useful in separating treatment effects in images or 380EX. Although comparable ratios were not available in 280EX data, the UV/blue ratio (F315/F420_280EX) was substantially reduced by UV-B exposure and was inversely related to total photosynthetic pigment content. These findings suggest that the red/green ratio (FImage, 380EX) and the UV/blue ratio (280EX) may be as useful as the blue/far-red ratio (380EX) reported previously in detection of UV-B stress. Furthermore, the results support the validity of the imaging technique as a non-destructive diagnostic tool for assessing UV-B stress damage in plants.     

Fluorescence emission spectra of plant leaves and plant constituents

Summary The UV-B radiation (e.g. 337 nm) induced blue fluorescence (BF) and red chlorophyll fluorescence spectra (RF) of green leaves from plants with different leaf structure were determined and the possible nature and candidates of the blue fluorescence emission investigated. The blue fluorescence BF is characterized by a main maximum in the 450 nm region and in most cases by a second maximum/shoulder in the 530 nm region. The latter has been termed green fluorescence GF. The red chlorophyll fluorescence RF, in turn, exhibits two maxima in the 690 and 730 nm region. In general, the intensity of BF, GF and RF emission is significantly higher in the lower than the upper leaf side. The ratio of BF to RF emission (F450/F690) seems to vary from plant species to plant species. BF and GF emission spectra appear to be a mixed signal composed of the fluorescence emission of several substances of the plant vacuole and cell wall, which may primarily arise in the epidermis. Leaves with removed epidermis and chlorophyll-free leaves, however, still exhibit a BF and GF emission. Candidates for the blue fluorescence emission ( max near 450 nm) are phenolic substances such as chlorogenic acid, caffeic acid, coumarins (aesculetin, scopoletin), stilbenes (t-stilbene, rhaponticin), the spectra of which are shown. GF emission ( max near 530 nm) seems to be caused by substances like the alkaloid berberine and quercetin. Riboflavine, NADPH and phyllohydroquinoneK ₁ seem to contribute little to the BF and GF emission as compared to the other plant compounds. Purified natural-carotene does not exhibit any blue fluorescence.     

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     

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.     

Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells I. Chlorophyll Accumulation and Phototransformation of Protochlorophyll(ide) in Callus Cells under Blue and Red light

A marked accumulation of chlorophyll was observed in calluscells of Nicotiana glutinosa when they were grown under bluelight, while under strong red light no chlorophyll accumulated.This blue light effect saturated at an intensity of about 500mW.m^–2. The effects of white, blue and red light on the transformationof protochlorophyll (ide) (Pchl) accumulated in dark-grown calluscells were studied by following the changes in the intensityof fluorescence emitted by Pchl and different forms of chlorophyll(ide) (Chi). Pchl with a fluorescence maximum at 633 nm (absorptionmaximum: 630 nm) decreased slowly, concomitant with an increasein Chl having a fluorescence maximum at 677 nm (absorption maximum:675 nm), which was subsequently transformed, independently oflight, to Chi with a fluorescence maximum at 683 nm (absorptionmaximum: 680 nm). Both blue and red light of low intensitieswere effective for the phototransformation, while red light,but not blue light, of high intensities caused significant destructionof Pchl. An action spectrum for this photodestruction showedthat the maximum destruction took place at 630 nm. White lightof high intensities was effective for the photoreduction withonly slight destruction of Pchl, suggesting that blue lightcounteracts the destructive effect of red light. At low temperatures,however, blue light as well as red light of low intensitiescaused photodestruction of Pchl. It was inferred that blue lightenhances a certain step or steps involved in the productionof a reductant required for the photoreduction of Pchl to Chl. (Received July 3, 1981; Accepted November 11, 1981)     

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.     

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.     

Far‐red light enhances photochemical efficiency in a wavelength‐dependent manner

Linear electron transport depends on balanced excitation of photosystem I and II. Far‐red light preferentially excites photosystem I (PSI) and can enhance the photosynthetic efficiency when combined with light that over‐excites photosystem II (PSII). The efficiency of different wavelengths of far‐red light exciting PSI was quantified by measuring the change in quantum yield of PSII (Φ_PSII) of lettuce (Lactuca sativa) under red/blue light with narrowband far‐red light added (from 678 to 752 nm, obtained using laser diodes). The Φ_PSII of lettuce increased with increasing wavelengths of added light from 678 to 703 nm, indicating longer wavelengths within this region are increasingly used more efficiently by PSI than by PSII. Adding 721 nm light resulted in similar Φ_PSII as adding 703 nm light, but Φ_PSII tended to decrease as wavelength increased from 721 to 731 nm, likely due to decreasing absorptance and low photon energy. Adding 752 nm light did not affect Φ_PSII. Leaf chlorophyll fluorescence light response measurements showed lettuce had higher Φ_PSII under halogen light (rich in far‐red) than under red/blue light (which over‐excites PSII). Far‐red light is more photosynthetically active than commonly believed, because of its synergistic interaction with light of shorter wavelengths.