Far-red Chlorophyll Fluorescence Research Articles

Tracking canopy chlorophyll fluorescence with a low-cost light emitting diode platform.

Chlorophyll fluorescence measured at the leaf scale through pulse amplitude modulation (PAM) has provided valuable insight into photosynthesis. At the canopy- and satellite-scale, solar-induced fluorescence (SIF) provides a method to estimate the photosynthetic activity of plants across spatiotemporal scales. However, retrieving SIF signal remotely requires instruments with high spectral resolution, making it difficult and often expensive to measure canopy-level steady-state chlorophyll fluorescence under natural sunlight. Considering this, we built a novel low-cost photodiode system that retrieves far-red chlorophyll fluorescence emission induced by a blue light emitting diode (LED) light source, for 2 h at night, above the canopy. Our objective was to determine if an active remote sensing-based night-time photodiode method could track changes in canopy-scale LED-induced chlorophyll fluorescence (LEDIF) during an imposed drought on a broadleaf evergreen shrub, Polygala myrtifolia. Far-red LEDIF (720-740 nm) was retrieved using low-cost photodiodes (LEDIFphotodiode) and validated against measurements from a hyperspectral spectroradiometer (LEDIFhyperspectral). To link the LEDIF signal with physiological drought response, we tracked stomatal conductance (gsw) using a porometer, two leaf-level vegetation indices-photochemical reflectance index and normalized difference vegetation index-to represent xanthophyll and chlorophyll pigment dynamics, respectively, and a PAM fluorimeter to measure photochemical and non-photochemical dynamics. Our results demonstrate a similar performance between the photodiode and hyperspectral retrievals of LEDIF (R2 = 0.77). Furthermore, LEDIFphotodiode closely tracked drought responses associated with a decrease in photochemical quenching (R2 = 0.69), Fv/Fm (R2 = 0.59) and leaf-level photochemical reflectance index (R2 = 0.59). Therefore, the low-cost LEDIFphotodiode approach has the potential to be a meaningful indicator of photosynthetic activity at spatial scales greater than an individual leaf and over time.

Detection of photosynthetic activity under cadmium stress by measurement of the red and far-red chlorophyll fluorescence

In this work it is shown that, with a new spectroscopic method based on ultraviolet light emitting diode-induced fluorescence spectroscopy (UV-LED IFS), the photosynthetic activity of an intact attached leaf can be measured in a non-destructive way within a few seconds. The effects of cadmium (Cd) toxicity on the photosynthetic activity of tomato plants (Solanum lycopersicum) were studied using various CdCl2 (cadmium chloride) concentrations. The Cd treatment decreases the photosynthetic activity, measured via a decline of the variable chlorophyll (chl) fluorescence decrease ratio (R Fd) values, calculated at 690 and 730 nm. The chl fluorescence ratios of intensity (FIR) and band area were also calculated. It was found that the R Fd(690) and R Fd(730) values decreased whereas the FIR increased from 1 μM Cd treatment and this behaviour is enhanced with the increasing of Cd concentrations. At 10 and 25 μM Cd, the decrease in R Fd values was associated with a strong decline in the chlorophyll-a + chlorophyll-b (chl-a + b). The chl-a + b decline could also be followed via an increase of the chl fluorescence ratio F 690/F 730. Our study demonstrates that the UV-LED IFS is a promising technique for early detection of Cd stress (from 1 μM) in tomato plants, before visual symptoms appear.

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment–protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715–730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.

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.

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.

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.

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.

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.