Chlorophyll Fluorescence Excitation Spectra Research Articles

Early optical detection of infection with brown rust in winter wheat by chlorophyll fluorescence excitation spectra

Puccinia recondita f. sp. tritici, the causative agent of brown rust disease is the reason for major yield losses in winter wheat plants (Triticum aestivum L.). Early detection of fungal infections would allow a specific fungicide application. Aim of the current study was to develop an automated and computer based device that differentiate infected and healthy plants non-invasively in an early stage of infection. To achieve the target an optical sensor device has been designed (“MultiDetExc”), which excites chlorophyll fluorescence in discrete wavelengths and detects the induced emissions. Wheat plants were infected artificially with brown rust in a climate chamber experiment to survey the capability of the instrument. The chlorophyll fluorescence excitation spectra of whole wheat plants were measured on several days after infection. As reference methods, HPLC and qPCR analysis were included in the current study to measure the polyphenol content of the leaves and the level of infection.The recently developed sensor device is an efficient technique to differentiate the infected and not infected wheat plants as soon as four days after inoculation. The measured fluorescence quotients correlate high positive with the polyphenol contents and the relative amount of fungal DNA. An untreated healthy control was measured parallelly, in order to associate the increased synthesis of polyphenols to the fungal infection definitely.

Light-induced accumulation of ortho-dihydroxylated flavonoids as non-destructively monitored by chlorophyll fluorescence excitation techniques

Chlorophyll fluorescence excitation techniques have been used in plant science for more than one decade to non-destructively estimate phenolic compounds in the epidermal cell layer. These techniques have been used here to evaluate the effect of different light intensities and spectral quality on the accumulation of ortho-dihydroxylated flavonoids in Phyllirea latifolia L., Myrtus communis L. and Ligustrum vulgare L. In a first experiment, chlorophyll fluorescence excitation spectra were measured (with a double arm optical fiber bundle connected to a spectrofluorimeter) on the adaxial and abaxial leaf surfaces of container-grown P. latifolia and M. communis exposed to 20% or 100% full sunlight. Differences in epidermal absorption spectra (referred to as epidermal absorption spectra throughout the paper) were then calculated from the relative chlorophyll fluorescence excitation spectra. This allowed comparing the content of UV-absorbing compounds between differentially irradiated leaves as well as between adaxial and abaxial epidermal layers. The absorption spectra were characterized by a band centered at 360–380 nm, which was greater in sun than in shade leaves and in the adaxial than in the abaxial surfaces, irrespective of species. Based upon HPLC-DAD and HPLC–MS analyses of leaf extracts and UV-spectral features of individual flavonoids, we conclude that quercetin, luteolin and myricetin derivatives were responsible for the observed light-induced changes in the spectral features of examined tissues. In a second experiment, we grew L. vulgare potted plants at 30% or 85% full sunlight in the presence or in the absence of UV radiation. We measured the absorbance characteristics at 370 nm of three leaf-pairs located in the apical portion of each shoot, using a portable fluorimetric sensor, the Multiplex ®. This allowed estimating, non-destructively, an index closely related to the concentration of epidermal flavonols and flavones having an ortho-dihydroxyl substitution in the B-ring. This index was greater in leaves growing at 85% than at 30% sunlight, irrespective of UV irradiance. When plants acclimated for 3 weeks to 30% sunlight, in the presence or in the absence of UV irradiance, were transferred to 85% sunlight, the flavonoid index exponentially increased, reaching a maximum within 10 days. On the whole, our experiments conclusively show that light-responsive flavonoids are mostly the dihydroxy B-ring-substituted structures. These flavonoids are the most effective, among the wide array of flavonoid structures, in preventing the generation and scavenging reactive oxygen species. As a consequence, we suggest that flavonoids do not merely serve as UV-screening agents, but behave as ROS-detoxifying agents in the mechanisms of photoprotection.

Effect of anthocyanins, carotenoids, and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis, assessment, modelling, and relevance to photoprotection

Whole apple fruit (Malus domestica Borkh.) widely differing in pigment content and composition has been examined by recording its chlorophyll fluorescence excitation and diffuse reflection spectra in the visible and near UV regions. Spectral bands sensitive to the pigment concentration have been identified, and linear models for non-destructive assessment of anthocyanins, carotenoids, and flavonols via chlorophyll fluorescence measurements are put forward. The adaptation of apple fruit to high light stress involves accumulation of these protective pigments, which absorb solar radiation in broad spectral ranges extending from UV to the green and, in anthocyanin-containing cultivars, to the red regions of the spectrum. In ripening apples the protective effect in the blue region could be attributed to extrathylakoid carotenoids. A simple model, which allows the simulation of chlorophyll fluorescence excitation spectra in the visible range and a quantitative evaluation of competitive absorption by anthocyanins, carotenoids, and flavonols, is described. Evidence is presented to support the view that anthocyanins, carotenoids, and flavonols play, in fruit with low-to-moderate pigment content, the role of internal traps (insofar as they compete with chlorophylls for the absorption of incident light in specific spectral bands), affecting thereby the shape of the chlorophyll fluorescence excitation spectrum.

Responses of epidermal phenolic compounds to light acclimation: In vivo qualitative and quantitative assessment using chlorophyll fluorescence excitation spectra in leaves of three woody species

Chlorophyll fluorescence (ChlF) excitation spectra were measured to assess the UV-sunscreen compounds accumulated in fully expanded leaves of three woody species belonging to different chemotaxons, (i.e. Morus nigra L., Prunus mahaleb L. and Lagerstroemia indica L.), grown in different light microclimates. The logarithm of the ratio of ChlF excitation spectra (logFER) between two leaves acclimated to different light microclimates was used to assess the difference in epidermal absorbance (EAbs). EAbs increased with increasing solar irradiance intercepted for the three species. This epidermal localisation of UV-absorbers was confirmed by the removal of the epidermis. It was possible to simulate EAbs as a linear combination of major phenolic compounds (Phen) identified in leaf methanol extracts by HPLC-DAD. Under UV-free radiation conditions, shaded leaves of M. nigra accumulated chlorogenic acid. Hydroxybenzoic acid (HBA) derivatives and hydroxycinnamic acid (HCA) derivatives greatly increased with increasing PAR irradiance under the low UV-B conditions found in the greenhouse. These traits were also observed for the HCA of the two other species. Flavonoid (FLAV) accumulation started under low UV-A irradiance, and became maximal in the adaxial epidermis of sun-exposed leaves outdoors. A decrease in the amount of HCA was observed concomitantly to the intense accumulation of FLAV for both leaf sides of the three species. Judging from the logFER, under low UV-B conditions, larger amounts of HCA are present in the epidermis in comparison to FLAV for the three species. Upon transition from the greenhouse to full sunlight outdoors, there was a decrease in leaf-soluble HCA that paralleled FLAV accumulation in reaction to increasing solar UV-B radiation in the three species. In M. nigra, that contains large amounts of HCA, the logFER analysis showed that this decrease occurred in the adaxial epidermis, whereas the abaxial epidermis, which is protected from direct UV-B radiation, continued to accumulate large amounts of HCA.

The use of chlorophyll fluorescence excitation spectra for the non‐destructive in situ assessment of UV‐absorbing compounds in leaves

AbstractIn this study a method was designed to assess non‐destructively the type of UV‐screening compounds present in the leaf epidermis. The method is based on the recording and calculation of the ratio of UV‐excitation spectra of chlorophyll fluorescence (FER) from the adaxial and abaxial sides of bifacial leaves, or from older and younger segments of monocotyledonous leaves. The logarithm of this ratio (logFER) matched the absorption spectrum of the UV‐absorbers present in the leaf, as confirmed by its overlap with the absorption spectrum of the methanolic extract of the leaf or of the isolated epidermis. By using the logFER approach, it was possible to demonstrate that the concentration but not the classes of compounds present in the epidermis that are responsible for UV‐screening is affected by the side and the age of the leaves. In contrast, measurements from the leaves of seven dicots and one monocot indicated large difference in the classes of these compounds between species. Finally, it was shown that the logFER in the UV is independent of the emission wavelength, and that the method can be used for quantitative measurements. This method expands to the spectral domain the use of ChlF for the estimation of the leaf epidermal transmittance.

Xanthophyll pigments in light-harvesting complex II in monomolecular layers: localisation, energy transfer and orientation

Monomolecular layers of the largest light-harvesting pigment-protein complex of Photosystem II (LHCII) were formed at the argon-water interface. The molecular area of the LHCII monomer in monomolecular layers determined from the isotherms of compression is found to be close to 14 nm 2, which corresponds well to the molecular dimensions of the protein evaluated on the basis of crystallographic studies. Monolayers of LHCII were deposited on a glass support by means of the Langmuir-Blodgett technique and subjected to spectroscopic studies: electronic absorption spectrophotometry and spectrofluorometry. The fluorescence excitation spectra of chlorophyll a in monolayers of LHCII were analysed using gaussian deconvolution. Comparison of the absorption and fluorescence excitation spectra enabled calculation of the rate of excitation energy transfer in the system. Excitation energy was found to be transferred to chlorophyll a from chlorophyll b with 97% efficiency, from neoxanthin with 85%, from lutein with 62% and from violaxanthin with at least 54% efficiency. The analysis of the position of the 0-0 absorption band of the xanthophylls revealed that neoxanthin is located in the same protein environment as lutein but in a different environment than violaxanthin. The analysis of fluorescence excitation spectra of chlorophyll a in LHCII, recorded with the excitation light beam polarised in two orthogonal directions, enabled the determination of the mean orientation angle of the accessory xanthophyll pigments with respect to the plane of the sample. The mean orientation of lutein found in this study (approx. 51°) corresponds well to the crystallographic data. Neoxanthin was found to adopt a similar orientation to lutein. The transition dipole moment of violaxanthin was found to form a mean angle of 71° with the axis spanning two polar regions of the protein, perpendicular to the plane of the monolayer, suggesting planar orientation of this pigment with respect to the plane of the thylakoid membrane. These experimentally determined xanthophyll orientations are discussed in terms of importance of peripheral xanthophyll pigments in supramolecular organisation of LHCII and the operation of the xanthophyll cycle within the thylakoid membrane.

Leaf Senescence of Postproduction Poinsettias in Low-light Stress

Photosynthetic light harvesting was investigated under low-light stress conditions relevant to the problem of interior longevity of potted ornamental plants. Comparisons of leaf pigment levels and chlorophyll fluorescence excitation spectra were made for `Gutbier V-10 Amy' poinsettia (Euphorbia pulcherrima Willd.), which has poor interior longevity, and `Eckespoint Lilo' poinsettia, which has superior interior longevity. The results show that `Eckespoint Lilo' had higher total chlorophyll content per leaf area and lower chlorophyll a: chlorophyll b ratio than `Gutbier V-10 Amy'. In low-light stress, `Eckespoint Lilo' retained its chlorophyll or even accumulated higher levels than in high light, while `Gutbier V-10 Amy' did not exhibit higher chlorophyll retention in low light. Both cultivars acclimatized to low-light stress by decreasing the chlorophyll a: chlorophyll b ratio, and this acclimatization was evident sooner in younger, outer-canopy leaves above the pinch than in older leaves below the pinch. Both cultivars also increased the chlorophyll: carotenoid ratio in low light. These changes in pigment composition, which were essentially structural changes, were reflected in functional changes in light harvesting, as assessed by measurements of chlorophyll fluorescence excitation spectra.

Specific solvation of chlorophyll b: A site-selection study at 5 K

The site-selection fluorescence excitation spectra of chlorophyll b were recorded in three different types of polar solvent host at 5 K. The influence of specific solvation of chlorophyll b on the S 1 state vibrational modes active in these spectra was studied in the high frequency vibrational region above 1000 cm −1. The nucleophilic solvation of chlorophylls ( i.e. one or two axial extra ligands at the central magnesium atom), unlike electrophilic interactions (hydrogen bonding), can be detected very successfully by the method used. The maximum down-shift of 20 – 30 cm −1 of the high characteristic vibrational frequencies in the CC stretching region estimated under electronic excitation of chlorophyll b is comparable with the effect of magnesium biligation and is explained in terms of an expansion of the tetrapyrrolic macrocycle core of the molecule. The broad-band fluorescence spectra of chlorophyll b in the same frozen solutions recorded at different laser excitation wavelengths are also presented and discussed.

Relationship between action spectra for chlorophyll a fluorescence and photosynthetic O2 evolution in algae

Relative excitation spectra of chlorophyll a fluorescence are shown to be very close to relative action spectra of photosystem II O2 evolution in seven micro- and macro-algae of five phyla. The conditions under which this correspondence should hold, based on theoretical considerations, and the applications of this correspondence to interpretation of fluorescence excitation spectra of chlorophyll a from natural popula- tions are discussed. The spectra presented are interpreted in relation to the light-harvesting pigments and their association with the two photosystems.

Photoadaptation in Marine Phytoplankton

The optical properties of marine phytoplankton were examined by measuring the absorption spectra and fluorescence excitation spectra of chlorophyll a for natural marine particles collected on glass fiber filters. Samples were collected at different depths from stations in temperate waters of the Southern California Bight and in polar waters of the Scotia and Ross Seas. At all stations, phytoplankton fluorescence excitation and absorption spectra changed systematically with depth and vertical stability of the water columns. In samples from deeper waters, both absorption and chlorophyll a fluorescence excitation spectra showed enhancement in the blue-to-green portion of the spectrum (470-560 nm) relative to that at 440 nm. Since similar changes in absorption and excitation were induced by incubating sea water samples at different light intensities, the changes in optical properties can be attributed to photoadaptation of the phytoplankton. The data indicate that in the natural populations studied, shade adaptation caused increases in the concentration of photosynthetic accessory pigments relative to chlorophyll a. These changes in cellular pigment composition were detectable within less than 1 day. Comparisons of absorption spectra with fluorescence excitation spectra indicate an apparent increase in the efficiency of sensitization of chlorophyll a fluorescence in the blue and green spectral regions for low light populations.