Far-red Fluorescence Research Articles

The properties of the chlorophyll a/b-binding proteins Lhca2 and Lhca3 studied in vivo using antisense inhibition.

The specific functions of the light-harvesting proteins Lhca2 and Lhca3 were studied in Arabidopsis ecotype Colombia antisense plants in which the proteins were individually repressed. The antisense effect was specific in each plant, but levels of Lhca proteins other than the targeted products were also affected. The contents of Lhca1 and Lhca4 were unaffected, but Lhca3 (in Lhca2-repressed plants) was almost completely depleted, and Lhca2 decreased to about 30% of wild-type levels in Lhca3-repressed plants. This suggests that the Lhca2 and Lhca3 proteins are in physical contact with each other and that they require each other for stability. Photosystem I fluorescence at 730 nm is thought to emanate from pigments bound to Lhca1 and Lhca4. However, fluorescence emission and excitation spectra suggest that Lhca2 and Lhca3, which fluoresce in vitro at 680 nm, also could contribute to far-red fluorescence in vivo. Spectral forms with absorption maxima at 695 and 715 nm, apparently with emission maxima at 702 and 735 nm, respectively, might be associated with Lhca2 and Lhca3.

Dual-excitation FLIDAR for the estimation of epidermal UV absorption in leaves and canopies

A new FLIDAR was designed for remote quantitative assessment of epidermal UV absorption of leaves and canopies from chlorophyll (Chl) fluorescence (ChlF) measurements. The dual-excitation fluorescence light detection and ranging (DE-FLIDAR) performs a dual excitation of the Chl present in leaves, in the UV (355 nm) and visible (532 nm) part of the spectrum, the latter being used as a reference excitation not absorbed by the epidermis. Therefore, the epidermal UV absorption of vegetation can be estimated from the Chl fluorescence excitation ratio (FER), ΦF(532)/ΦF(355). Thanks to the alternated excitation by the DE-FLIDAR, the FER is immune to natural conditions in field, such as light-induced variable ChlF and leaf movement (variation of the angle of excitation). The DE-FLIDAR was used to investigate the presence of UV-absorbing compounds in individual leaves and canopies of different plant species, tobacco, pea, barley and wheat. The FER was much larger in outdoor-grown plants, indicating an accumulation of UV-absorbing compounds. We also analysed the epidermal UV absorption of the adaxial and abaxial side of tobacco leaves of different age. The logarithm of the FER showed a good agreement with the absorbance of methanolic extracts obtained from the same leaves. The presented DE-FLIDAR can perform up to three simultaneous fluorescence measurements; therefore, we could compare blue fluorescence (BR) to the epidermal UV absorption. In addition, a dual ratio, the red fluorescence (RF) to far-red fluorescence (FRF) emission ratio, excited at 355 and 532 nm, was shown to be linearly dependent on the Chl content. A mathematical model of leaf absorption and fluorescence, based on the Beer–Lambert's law, was developed to describe and analyse the fluorescence signatures obtained with the DE-FLIDAR.

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.

Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence

A novel gene for advanced red-shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP-like protein drFP583 with an emission maximum at 583 nm. Using the remarkable similarity of the drFP583 and dsFP593 genes, we performed a ‘shuffling’ procedure to generate a pool of mutants consisting of various combinations of parts of both genes. One ‘hybrid gene’ was chosen for subsequent random mutagenesis, which resulted in a mutant variant with a uniquely red-shifted emission maximum at 616 nm.

A passive two-band sensor of sunlight-excited plant fluorescence

We have designed and built a passive remote sensor of sunlight-excited chlorophyll fluorescence (U.S. Patent No. 5,567,947, Oct. 22, 1996) which provides for the real-time, in situ sensing of photosynthetic activity in plants. This sensor, which operates as a Fraunhofer line discriminator, detects light at the cores of the lines comprising the atmospheric oxygen A and B bands, centered at 762 and 688 nm, respectively. These bands also correspond to wavelengths in the far-red and red chlorophyll fluorescence bands. The sensor is based on an induced fluorescence approach; as light collected from fluorescing plants is passed through a low-pressure cell containing oxygen, the oxygen absorbs the energy and subsequently reemits photons which are then detected by a photomultiplier tube. Since the oxygen in the cell absorbs light at the same wavelengths that have been strongly absorbed by the oxygen in the atmosphere, the response to incident sunlight is minimal. This mode of measurement is limited to target plants sufficiently close in range that the plants’ fluorescence is not itself appreciably absorbed by atmospheric oxygen (∼200 m). In vivo measurements of fluorescence in the 760 and 690 nm bands of vegetation in full sunlight are also presented. Measurements of plant fluorescence at the single-plant canopy level were obtained from greenhouse-grown bean plants subjected to a range of nitrogen treatments. The ratio of the fluorescence obtained from the two measurement bands showed statistically significant variation with respect to nitrogen treatments.

Contribution of Chlorophyll Fluorescence to the Reflectance of Leaves in Stressed Plants as Determined with the VIRAF-Spectrometer

Reflectance spectra allow the early detection of stressors causing differences in pigment content as well as changes of leaf tissue structure and photosynthetic activity. The reflectance decreased with increasing Chl content in greening bean leaves. In stressed leaves, in turn, the reflectance increased with decreasing Chl content. This also caused a shift of the red reflection rise (“red edge”) to shorter wavelengths (“blue shift”) associated with a blue shift of the inflection point of the red edge. The contribution of the red and far-red Chl fluorescence to the reflectance signal at the red edge of the spectrum and the shift of the wavelength position of the inflection point are demonstrated and discussed

Response of thein vivochlorophyll fluorescence spectrum to environmental factors and laser excitation wavelength

The laser-induced chlorophyll (Chl) fluorescence spectrum in vivo at the steady state was investigated as a function of the leaf temperature between 25 and at low light intensity . The ratio between the red and far-red fluorescence bands, F685/F730, was seen to decrease with decreasing leaf temperature, while the fluorescence intensity at both the 685 and 730 nm peaks increased going from 25 to . This behaviour appears to be the same in chilling-sensitive plants ( Lycopersicon esculentum and Phaseolus vulgaris) as well as in chilling-tolerant species ( Pisum sativum and Vicia faba). Under high light intensity , at controlled temperature, F685/F730 as well as the total fluorescence intensity was seen to decrease with irradiation time. Since photosystem I (PSI) contributes to the F730 emission band only, while photosystem II (PSII) contributes to both F685 and F730, the above results can be explained as being due to the change with light intensity and temperature of quenching processes that affect PSII more than PSI. Changing the light intensity impinging on the leaf, the total Chl fluorescence increases with increasing light intensity to a maximum reached at about , then it decreases to values close to the dark level of fluorescence . The Chl fluorescence spectrum was seen to vary in shape by changing the excitation wavelength. The well known process of Chl fluorescence reabsorption is responsible for the decrease in the F685/F730 going from less (440 nm) to more (635 nm) penetrating excitation light. With UV excitation at 337 nm, an unexpected lower value for F685/F730 with respect to blue excitation was found.

Principles and characteristics of multi-colour fluorescence imaging of plants

Green plants illuminated with UV-radiation emit a blue and green fluorescence as well as a red and farred chlorophyll fluorescence with maxima near 440, 520, 690 and 740 nm, respectively. In contrast to point data measurements, the establishment of a high spatial resolution multi-colour fluorescence imaging system, the Karlsruhe/Strasbourg Fluorescence Imaging System (FIS), permits to simultaneously screen several hundred picture elements (pixels) of the leaf in the four fluorescence bands of plants, and opens new possibilities for physiology measurements and early stress detection. This review describes the principles and particular characteristics a) of the plants' fluorescence signatures, b) of fluorescence imaging in general and c) of the Karlsruhe/Strasbourg high resolution multi-colour fluorescence imaging system FIS. The origin of the fluorescence signals in the blue and green (cinnamic acids and other plant phenolics), the red and far-red (chlorophyll a) and the factors influencing these fluorescence signatures are explained. By means of an intensified video camera, fluorescence images with several hundred picture elements are taken, not only of the red and far-red Chl fluorescence, but also of the blue and green plant fluorescence, as multicolour images. Via computer-aided data processing one obtains false colour images not only of the fluorescence intensity, but also of the fluorescence ratios of whole leaves. The fluorescence ratios blue/red (F440/ F690) and blue/far-red (F440/F740) proved to be very sensitive early stress and strain indicators of plants. The fluorescence ratio F690/F740 is an indicator of the in situ Chl content. By fluorescence imaging of the variable Chl fluorescence (Rfd-values) one can also simultaneously measure the potential photosynthetic activity of all points of a leaf. At short-term stress, the fluorescence ratio blue/green (F440/F520) is relatively stable, but changes at long-term stress, often by an increase of the green fluorescence emission. Due to the high statistic confidence (large pixel numbers) and the excellent possibilities to detect specific distribution gradients and local irregularities in fluorescence emission and fluorescence ratios over the leaf area, fluorescence imaging is a superior means for early stress and damage detection in plants.

Fluorescence Counter-Staining of Cell Nuclear DNA for Multi-Color Laser Confocal Microscopy.

Twenty-one nucleic acid-specific fluorochromes were examined for use in cell nuclear counter-staining of specimens observed under a laser confocal microscope equipped with a Krypton/argon laser tube. Six green, three red, and two far-red fluorochromes gave sufficient fluorescence. Among them, SYBR Green I, Pico Green, SYTOX Green, YO-PRO-3, and TO-PRO-3 specifically stained the cell nuclear DNA. Other fluorochromes except POPO-3 stained the cell nuclear DNA and co-stained the cytoplasmic RNA. Conversely, POPO-3 specifically stained the cytoplasmic and nucleolar RNA. Anti-bleaching reagents (PPDA and DABCO) effectively prevented the photobleaching of SYBR Green I, Pico Green, YO-PRO-3, and TO-PRO-3, whereas DABCO accelerated the photobleaching of SYTOX Green. Taking into account the characteristics of these fluorochromes, SYBR Green I, YO-PRO-3, and TO-PRO-3 were best suited for cell nuclear counter-staining as green, red, and far-red fluorochromes, respectively. Especially, TO-PRO-3 seems to be the most useful because its far-red fluorescence enables three-color fluorescence staining by combination with immunofluorescence labeling with fluorescein and rhodamine derivatives.

Cell wall bound ferulic acid, the major substance of the blue-green fluorescence emission of plants

Summary When excited by UV-A radiation (e.g. N 2 -laser 337 nm), leaves of green plants exhibit, in addition to the red and far-red chlorophyll fluorescence, a genuine blue-green fluorescence emission with a maximum near 440–460 nm and a lower shoulder near 520–530 nm. Members of the Poaceae (monocotyledonous plants), e.g. maize ( Zea mays L.), wheat ( Triticum aestivum L.) or oat ( Auma sativa L.), possessed a much higher blue-green fluorescence emission than leaves of dicotyledonous plants, such as spinach ( Spinacia oleracea L.), tobacco ( Nicotiana tabacum L.), sunflower ( Helianthus annuus L.), foxtail ( Amaranthus caudatus L.) or purslane ( Portulaca oleracea L.). In the measured dicotyledonous plants, the blue-green fluorescence was in a similar range or considerably lower than the red chlorophyll fluorescence. In contrast, in Poaceae and other monocotyledonous plants, the blue-green fluorescence was generally several times higher than the red chlorophyll fluorescence. By alkaline hydrolysis of cell walls it was shown that the members of Poaceae exhibit a several times higher content of the blue-green fluorescent ferulic acid, both on leaf area and on a dry weight basis, than dicotyledonous plants, such as spinach, foxtail and purslane. Ferulic acid, being covalendy bound to the cell wall, appears to be the main emitter of the blue-green fluorescence of leaves which has been documented by several complementary observations. Other cell wall bound cinnamic acids (caffeic acid, p-coumaric acid), which are present in some plants in low amounts, contribute very little to the overall blue-green fluorescence emission of leaves. This also applies to the extractable flavonoids and cinnamic acids of the vacuole. The high fluorescence yield of ferulic acid containing cell walls is also documented by fluorescence excitation spectra of isolated, dried cell walls before and after alkaline hydrolysis of ferulic acid. Plants which do not possess ferulic acid in their cell walls, such as sunflower, pumpkin ( Cucurbita fici-folia L.) or tobacco, exhibit only a very faint blue-green fluorescence emission. Outdoor plants contained much higher levels of extractable flavonoids and cinnamic acids than greenhouse plants, yet the blue-green fluorescence was hardly modified indicating that the soluble flavonoids and cinnamic acids of the vacuole do not or only very little contribute to the blue-green fluorescence emission of plants.

Curve-fit analysis of chlorophyll fluorescence spectra: Application to nutrient stress detection in sunflower

The in vivo laser-induced chlorophyll fluorescence (LICF) spectra of healthy and nutrient-deficient sunflower plants were measured on a Jobin Yvon monochromator with HeNe laser excitation. To correctly determine the peak center and to evaluate the relative contributions of the bands in the total fluorescence spectrum, the steady state LICF spectra were analyzed with a nonlinear iterative procedure using Gaussian, Lorentzian, Pearson, Voigt, and exponential Gaussian spectral functions. It was observed that curve fitting performed by using two Gaussian peaks centered at 690 and 730 nm usually fits well to the chlorophyll fluorescence spectra. After curve fitting, the mean peak centers of the red and far-red chlorophyll bands of control plants were observed at 688.2 and 725.4 nm, respectively. A blue shift of as much as 9 nm in the peak position of the far-red band was observed with nutrient stress, whereas the shift in position of the red band was only of the order of a few nanometers. Further, the width at half maximum of the far-red band was found to increase by as much as 20 nm with nutrient stress. Curve fitting could thus separate out the red and far-red fluorescence spectra from a pair of normally distributed curves centered at 690 and 730 nm wavelengths, thereby differentiating the effects due to reabsorption from those due directly to changes in photosynthetic electron transport. The F690F730 fluorescence intensity ratio obtained from curve-fitted parameters was found to be more sensitive to plant stress than were fluorescence values alone. Results indicate that a curve-fitting analysis of LICF spectra using Gaussian spectral functions is a very useful and sensitive method of evaluating spectral features from a statistical point of view and for accurate determination of contributions from constituent bands in the whole leaf fluorescence spectrum.

DNA staining for fluorescence and laser confocal microscopy.

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

Fluorescence Imaging of Water and Temperature Stress in Plant Leaves

Summary Fluorescence images of leaves from tobacco plants (green wild type and aurea mutant) were determinedin the blue (F440), green (F520), red (F690) and far-red region (F740), and also expressed as fluorescence ratio images. Under long-term water stress tobacco plants initially showed constant ratios of the blue to red fluorescence (F440/F690) and the blue to far-red fluorescence (F440/F740). Below a distinct threshold in water content (84° in green and 88° in aurea tobacco), however, a linear increase of the fluorescence ratios blue/red and blue/far-red was observed. This was due to a distinct increase in the bluegreen fluorescence emission, whereas the red and far-red chlorophyll fluorescence increased to a lower proportion. These changes in fluorescence ratios could easily be monitored by high resolution fluorescence imaging of whole leaves. For each point of the leaf, the fluorescence ratio can be read from the fluorescence ratio images of the leaves. In contrast, a short-term heat plus water stress in green tobacco plants was very fast detected via fluorescence imaging as a significant increase of red and far-red chlorophyll fluorescence emission (F690 and F740) on the leaf rim, whereas the central part of the leaf still exhibited the regular fluorescence signatures of photosynthetically active leaves. A combined outdoor stress (light, heat and water stress) at a dry sunny summer period was detected in Rhododendron by fluorescence imaging due to a much reduced red and far-red chlorophyll fluorescence. The latter was caused by UV-absorbing substances (e.g. flavonols) which accumulated primarily in the epidermis of these stressed leaves. These compounds seemed to act as UV-radiation filter, thus reducing the amount of the UV-excitation radiation, which could penetrate the mesophyll and which resulted in a reduced chlorophyll fluorescence excitation and emission. These results demonstrate that fluorescence imaging of leaves in the blue, green, red and far-red emission bands is an excellent tool for an early stress detection in plants, which is much superior to the hitherto applied spectral point data measurements.

Detection of Vegetation Stress Via a New High Resolution Fluorescence Imaging System

The UV-laser ( λ 355 nm) induced fluorescence emission spectra of green leaves comprise the blue(F440) and green (F520) fluorescence bands as well as the red (F690) and far-red (F740) chlorophyll fluorescence emission bands. Based on the four UV-laser induced fluorescence bands blue, green, red and farred a high resolution fluorescence imaging system was established, which allows a fast and large scale screening of fluorescence gradients and local disturbances in fluorescence emission over the whole leaf surface. The new imaging method not only permits to screen leaves by means of images in four fluorescence bands (LIF images) but, in addition, via images of the fluorescence ratios blue/red (F440/F690), blue/farred (F440/F740), the chlorophyll fluorescence ratio red/far-red (F690/F740) and the ratio blue/green (F440/F520) (LIF ratio images). By fluorescence imaging we could prove that in aurea tobacco the major part of the leaves' blue and green fluorescence is emitted from the main and side leave veins, whereas the major part of the leaves' red and far-red chlorophyll fluorescence is emitted from the vein-free leaf regions, which also have the highest chlorophyll content. A smaller proportion of the aurea tobacco leaves' blue-green fluorescence emission is derived from the cell walls of epidermis cells. The fluorescence ratios blue/red and blue/far-red are very sensitive to environmental changes, and thus permit early stress and strain detection in plants, and the evaluation of damage to the photosynthetic apparatus. Via monitoring the increase in chlorophyll fluorescence LIF images allow to detect differences in the time-dependent uptake of diuron and the progressing inhibition of photosynthetic electron transport in the treated leaf part. The novel fluorescence imaging technique sets a new dimension for early stress detection in the photosynthetic apparatus and in plants. It has many advantages over the previously applied point-data measurements of selected leaf points using conventional spectrofluorometers. The new fluorescence imaging system proved to be very suitable for remote sensing of plants in the near distance, and can be further developed for far distance remote sensing of the state of health of terrestrial vegetation. Some examples in the many possible ways of computer-aided fluorescence data processing (formation of different fluorescence ratios, screening of fluorescence profiles, histogramme plotting) are indicated.

Differences in Fluorescence Excitation Spectra of Leaves between Stressed and Non-Stressed Plants

Summary Using a Perkin-Elmer fluorometer the fluorescence excitation spectra of the four main fluorescencebands of plants (blue, green, red and far-red) were determined in the UV-region between 250 to 400 nm. The results provide valuable information on the most suitable wavelength for fluorescence excitation which is different for the blue and green fluorescence from that of the red and far-red chlorophyll fluorescence. The differing fluorescence yield in dependence of the excitation wavelength can be quantified by the fluorescence ratio blue/red (F450/F690) and blue/far-red (F450/F735), as well as red/far-red (F690/ F735) and blue/green (F450/F530). The differential values of the fluorescence ratios, as found for the usually applied laser-excitation wavelengths of 308, 337, 355 and 397nm, are contrasted. The study demonstrates that fluorescence emission spectra of leaves can successfully be applied for stress detection in plants. The fluorescence ratios blue/red (F450/F690) and blue/far-red (F450/F735) proved to be the most sensitive and best suited stress indicators. For simultaneous excitation of the blue-green fluorescence and the red+far-red chlorophyll fluorescence an excitation wavelength between 355 and 390 nm is recommended. Via fluorescence excitation spectra it was also proved that sun leaves, outdoor plants and highlight treated plants are better protected against UV-A and UV-B than shade leaves, greenhouse plants or low-light plants. The transformation in epidermis cells of UV-radiation into blue and green fluorescence, which can be used by mesophyll cells for photosynthetic quantum conversion, is a new protection mechanism of plants and their photosynthetic apparatus against UV-radiation.

Comparison of chlorophyll far-red and rea fluorescence excitation spectra with photo-synthetic oxygen action spectra for photo-system II in algae

Comparison of chlorophyll far-red and rea fluorescence excitation spectra with photo-synthetic oxygen action spectra for photo-system II in algae

Chloroplast fluorescence of C4 plants

The precise spectral responses of infrared color film permit a rapid qualitative detection of the in situ fluorescence properties of chloroplasts in C4 plants. Fluorescing mesophyll and granal bundle-sheath chloroplasts appear yellow, indicating a predominance of far-red over infrared fluorescence. In contrast, fluorescing agranal bundle-sheath chloroplasts appear red, indicating a predominance of infrared over far-red fluorescence. There is a strong positive correlation between lamellar appression and the ratio of far-red to infrared fluorescence as recorded by infrared color film. The red versus yellow color of bundle-sheath chloroplast infrared photographs is used to separate the 18 species of C4 plants and 2 species of C3 plants examined into two basic groups. This separation is compatible with a separation based on lamellar ultrastructure of bundle-sheath chloroplasts.