Chl Fluorescence Ratio Research Articles

Multi-colour fluorescence imaging of photosynthetic activity and plant stress

Imaging the four fluorescence bands of leaves, the red (F690) and far-red (F740) chlorophyll (Chl) fluorescence as well as the blue (F440) and green (F520) fluorescence of leaves and the corresponding fluorescence ratios is a fast and excellent nondestructive technique to detect the photosynthetic activity and capacity of leaves, of gradients over the leaf area as well as the effect of various strain and stress parameters on plants. This review primarily deals with the first and pioneering multi-colour fluorescence imaging results obtained since the mid-1990s in a cooperation with French colleagues in Strasbourg and in my laboratory in Karlsruhe. Together we introduced not only the joint imaging of the red and far-red Chl fluorescence but also of the blue and green fluorescence of leaves. The two instrumental setups composed for this purpose were (1) the Karlsruhe-Strasbourg UV-Laser Fluorescence Imaging System (Laser-FIS) and (2) the Karlsruhe Flash-Light Fluorescence Imaging System (FL-FIS). Essential results obtained with these instruments are summarized as well as the basic principles and characteristics of multi-colour fluorescence imaging. The great advantage of fluorescence imaging is that the fluorescence yield in the four fluorescence bands is sensed of several thousand up to 200,000 pixels per leaf area in one image. The multi-colour FIS technique allows to sense many physiological parameters and stress effects in plants at an early stage before a damage of leaves is visually detectable. Various examples of plant stress detection by the multi-colour FIS technique are given. Via imaging the Chl fluorescence ratio F690/F740 it is even possible to determine the Chl content of leaves. The FIS technique also allows to follow the successive uptake of diuron and loss of photosynthetic function and to screen the ripening of apples during storage. Particularly meaningful and of high statistical relevance are the fluorescence ratio images red/far-red (F690/F740), blue/red (F440/F690), and blue/green (F440/F520) as well as images of the fluorescence decrease ratio RFd, which is an indicator of the net CO2 assimilation rates of leaves.

Characterisation of estuarine intertidal macroalgae by laser-induced fluorescence

The article reports the application of laser-induced fluorescence (LIF) for the assessment of macroalgae communities of estuarine intertidal areas. The method was applied for the characterisation of fifteen intertidal macroalgae species of the Tagus estuary, Portugal, and adjacent coastal area. Three bands characterised the LIF spectra of red macroalgae with emission maxima in the ranges 577–583 nm, 621–642 nm and 705–731 nm. Green and brown macroalgae showed one emission maximum in the red region (687–690 nm) and/or one in the far-red region (726–732 nm). Characteristics of LIF emission spectra were determined by differences in the main fluorescing pigments: phycoerythrin, phycocyanin and chlorophyll a (Chl a). In the green and brown macroalgae groups, the relative significance of the two emission maxima seems to be related to the thickness of the photosynthetic layer. In thick macroalgae, like Codium tomentosum or Fucus vesiculosus, the contribution of the far-red emission fluorescence peak was more significant, most probably due to re-absorption of the emitted red Chl a fluorescence within the dense photosynthetic layer. Similarly, an increase in the number of layers of the thin-blade green macroalgae Ulva rigida caused a shift to longer wavelengths of the red emission maximum and the development of a fluorescence peak at the far-red region. Water loss from Ulva's algal tissue also led to a decrease in the red/far-red Chl fluorescence ratio (F685/F735), indicating an increase in the density of chloroplasts in the shrinking macroalgal tissue during low tide exposure.

Detection of mosaic virus disease in cassava plants by sunlight-induced fluorescence imaging: a pilot study for proximal sensing

Cassava mosaic disease (CMD) is a prominent virus infection that causes considerable crop damage and yield reduction. Early detection of crop damage by remote sensing could be a useful tool for initiating remedial measures to reduce further crop damage. This article presents a non-destructive method for detection and classification of CMD infection, based on the red:far-red chlorophyll (chl) fluorescence image ratio. This pilot study was carried out in 14 varieties of potted cassava plants (Manihot esculenta Crantz) with a multispectral imaging system (MSIS) consisting of an electron multiplying charge coupled device (EMCCD) camera. Sunlight-induced chl fluorescence (SICF) images of plant leaves were recorded using the MSIS at the Fraunhofer lines of O2-B at 687 nm and O2-A at 759.5 nm and their off-lines at 684 and 757.5 nm. The recorded images were analysed using the Fraunhofer line discrimination (FLD) technique to extract the SICF from the solar reflectance in the recorded images. The chl fluorescence image ratio (red:far-red, F687:F760) was computed and correlated with the laser-induced chl fluorescence (LICF) ratio (F685:F735) determined by point monitoring, chl content variation, and the net photosynthetic rate (Pn). The scatter plot of the F687:F760 image ratio showed good discrimination between different levels of CMD infection as evidenced by the high sensitivity and specificity values. It is observed that the fluorescence image ratio (F687:F760) has a good correlation with Pn (coefficient of determination (R2) = 0.85), chl content (R2 = 0.82), and the LICF ratio (F685:F735) (R2 = 0.80), thereby highlighting the potential of the SICF image ratio in the discrimination of CMD infection. The results clearly indicate that changes in the red:far-red fluorescence image ratio due to CMD stress can easily be detected at an early stage and the technique has great potential for monitoring the health of crops and vegetation from proximal sensing platforms.

Chlorophyll fluorescence kinetics, photosynthetic activity, and pigment composition of blue-shade and half-shade leaves as compared to sun and shade leaves of different trees

The chlorophyll (Chl) fluorescence induction kinetics, net photosynthetic CO2 fixation rates P N, and composition of photosynthetic pigments of differently light exposed leaves of several trees were comparatively measured to determine the differences in photosynthetic activity and pigment adaptation of leaves. The functional measurements were carried out with sun, half-shade and shade leaves of seven different trees species. These were: Acer platanoides L., Ginkgo biloba L., Fagus sylvatica L., Platanus x acerifolia Willd., Populus nigra L., Quercus robur L., Tilia cordata Mill. In three cases (beech, ginkgo, and oak), we compared the Chl fluorescence kinetics and photosynthetic rates of blue-shade leaves of the north tree crown receiving only blue sky light but no direct sunlight with that of sun leaves. In these cases, we also determined in detail the pigment composition of all four leaf types. In addition, we determined the quantum irradiance and spectral irradiance of direct sunlight, blue skylight as well as the irradiance in half shade and full shade. The results indicate that sun leaves possess significantly higher mean values for the net CO2 fixation rates P N (7.8-10.7μmol CO2m(-2)s(-1) leaf area) and the Chl fluorescence ratio R Fd (3.85-4.46) as compared to shade leaves (mean P N of 2.6-3.8μmol CO2m(-2)s(-1) leaf area.; mean R Fd of 1.94-2.56). Sun leaves also exhibit higher mean values for the pigment ratio Chl a/b (3.14-3.31) and considerably lower values for the weight ratio total chlorophylls to total carotenoids, (a+b)/(x+c), (4.07-4.25) as compared to shade leaves (Chl a/b 2.62-2.72) and (a+b)/(x+c) of 5.18-5.54. Blue-shade and half-shade leaves have an intermediate position between sun and shade leaves in all investigated parameters including the ratio F v/F o (maximum quantum yield of PS2 photochemistry) and are significantly different from sun and shade leaves but could not be differentiated from each other. The mean values of the Chl fluorescence decrease ratio R Fd of blue-shade and half-shade leaves fit well into the strong linear correlation with the net photosynthetic rates P N of sun and shade leaves, thus unequivocally indicating that the determination of the Chl fluorescence decrease ratio R Fd is a fast and indirect measurement of the photosynthetic activity of leaves. The investigations clearly demonstrate that the photosynthetic capacity and pigment composition of leaves and chloroplasts strongly depend on the amounts and quality of light received by the leaves.

Effect of 24-epibrassinolide on drought stress-induced changes in Chorispora bungeana

Brassinosteroids (BRs) have been proposed to increase the resistance of plants to drought stress. The effect of foliar application of 0.1 μM 24-epibrassinolide (EBR) on chlorophyll (Chl) content, photosystem 2 (PS 2) photochemistry, membrane permeability, lipid peroxidation, relative water content (RWC), proline content, and the antioxidant system in drought-stressed Chorispora bungeana plants was investigated. The results showed that polyethylene glycol (PEG) induced water stress decreased RWC, Chl content and variable to maximum Chl fluorescence ratio (Fv/Fm) less in plants pretreated with EBR than in non-pretreated plants. In addition, lipid peroxidation, measured in terms of malondialdehyde content, membrane permeability and proline content in drought-stressed plants were less increased in EBR pretreated plants, while antioxidative enzyme activities and reduced ascorbate and glutathione contents were more increased in EBR pretreated than in non-pretreated plants. These results suggested that EBR could improve plant growth under drought stress

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.

Responses of the resurrection plant Haberlea rhodopensis to high irradiance

The effect of high irradiance (HI) during desiccation and subsequent rehydration of the homoiochlorophyllous desiccation-tolerant shade plant Haberlea rhodopensis was investigated. Plants were irradiated with a high quantum fluence rate (HI; 350 µmol m−2 s−1 compared to ca. 30 µmol m−2 s−1 at the natural rock habitat below trees) and subjected either to fast desiccation (tufts dehydrated with naturally occurring thin soil layers) or slow desiccation (tufts planted in pots in peat-soil dehydrated by withholding irrigation). Leaf water content was 5 % of the control after 4 d of fast and 19 d of slow desiccation. Haberlea was very sensitive to HI under all conditions. After 19 d at HI, even in well-watered plants there was a strong reduction of rates of net photosynthesis and transpiration, contents of chlorophyll (Chl) and carotenoids, as well as photosystem 2 activity (detected by the Chl fluorescence ratio RFd). Simultaneously, the blue/red and green/red fluorescence ratios increased considerably suggesting increased synthesis of polyphenolic compounds. Desiccation of plants in HI induced irreversible changes in the photosynthetic apparatus and leaves did not recover after rehydration regardless of fast or slow desiccation. Only young leaves survived desiccation.

Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves

Various approaches to understand and make use of the variable chlorophyll (Chl) fluorescence emission spectrum and fluorescence ratio are reviewed. The Chl fluorescence of leaves consists of two maxima in the red (near 685-690 nm), and far-red region (near 730-740 nm). The intensity and shape of the Chl fluorescence emission spectrum of leaves at room temperature are primarily dependent on the concentration of the fluorophore Chl a, and to a lower degree also on the leaf structure, the photosynthetic activity, and the leaf's optical properties. The latter determine the penetration of excitation light into the leaf as well as the emission of Chl fluorescence from different depths of the leaf. Due to the re-absorption mainly of the red Chl fluorescence band emitted inside the leaf, the ratio between the red and the far-red Chl fluorescence maxima (near 690 and 730-740 nm, respectively), e.g., as F690/F735, decreases with increasing Chl content in a curvilinear relationship and is a good inverse indicator of the Chl content of the leaf tissue, e.g., before and after stress events. The Chl fluorescence ratio of leaves can be applied for Chl determinations in basic photosynthesis research, agriculture, horticulture, and forestry. It can be used to assess changes of the photosynthetic apparatus, developmental processes of leaves, state of health, stress events, stress tolerance, and also to detect diseases or N-deficiency of plants.

The Effect of Decreasing Temperature up to Chilling Values on the in vivo F685/F735 Chlorophyll Fluorescence Ratio in Phaseolus vulgaris and Pisum sativum: The Role of the Photosystem I Contribution to the 735 nm Fluorescence Band ¶

The effect of leaf temperature (T), between 23 and 4°C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 μE m−2 s−1) and low (30–35 μE m−2 s−1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1–state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence lifetime measurements support this interpretation. At a critical temperature (about 6°C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spillover from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

Photosynthetic response of different pea cultivars to low and high temperature treatments

The thermo-sensitivity of two new pea (Pisum sativum L.) cultivars-Afila (mutant in the gene transforming leaves into mustaches) and Ranen (mutant for early ripening)-as compared to the control cultivar Pleven-4 to either low (4 °C, T4) or high temperature (38 °C, T38) was investigated by means of chlorophyll (Chl) fluorescence kinetics. The low temperature treatment decreased the photosynthetic activity, measured via a decline of the Chl fluorescence decrease ratios RFd690 and RFd735, and this was mainly due to a decline of the Chl fluorescence decrease parameter Fd and maximum Chl fluorescence Fm. In the new cv. Ranen the RFd ratios at first decreased and increased again after 24-h exposure to 4 °C, indicating its good acclimation ability to low temperature. The cold-induced changes in the photosynthetic performance of all cultivars were reversed after transferring plants back to 23 °C for 48 h. In the Chl and carotenoid (Car) contents no or little changes occurred during the T4 treatment, except for a slight but clear increase of the ratio Chl a/b and a decrease in the ratio Chl/Car. In contrast to this, the T38 treatment for 72 h decreased the RFd ratios more strongly than the T4 exposure did. In fact, an irreversible injury of the photosynthetic apparatus was caused in the control pea cv. Pleven-4 by a 48-h T38 exposure and for the new cv. Afila after a 72-h T38 exposure. In contrast, the cv. Ranen was less and little sensitive to the T38 exposure. In the heat-sensitive cvs. Pleven-4 and Afila, the decrease in RFd values at T38 was associated with a strong decline of the Chl a+b and total Car contents. The Chl a+b decline could also be followed via an increase of the Chl fluorescence ratio F690/F735. Parallel to this, a strong decline of Chl a/b from ca. 3.0 (range 2.85-3.15) to ca. 1.9 (range 1.85-1.95) occurred indicating a preferential decline of the Chl a-pigment proteins but not of the Chl a/b-pigment protein LHC2. In the relatively heat-tolerant cv. Ranen, however, the ratio Chl a/b declined only partially. After the T4 treatment the stress adaptation index Ap was higher in cv. Ranen than in controls and reached in heat-treated Ranen plants almost the starting value indicating a cold and heat stress hardening of the treated plants. The Chl fluorescence parameters and pigment contents were influenced by T38 and T4 treatments in various ways indicating that the mechanisms of low and high temperature injury of the photosynthetic apparatus are different. The new cv. Ranen exhibited a cross tolerance showing a fairly good acclimation ability to both T4 and T38, hence it is a very suitable plant for outdoor growth and for clarification of the acclimation mechanisms to unfavourable temperatures.

Effects of abscisic acid or benzyladenine on pigment contents, chlorophyll fluorescence, and chloroplast ultrastructure during water stress and after rehydration

With the aim to contribute to the elucidation of the role of phytohormones in response of plants to adverse environmental conditions, seedlings of Phaseolus vulgaris, Nicotiana tabacum, Beta vulgaris, and Zea mays were supplied with water, 100 µM abscisic acid (ABA), or 10 µM N6-benzyladenine (BA) immediately before imposition of water stress (WS). In all four species, contents of chlorophylls (Chls) and carotenoids were markedly decreased during WS and after rehydration only in plants pre-treated with water but not in those pre-treated with ABA or BA. Contents of pigments of xanthophyll cycle increased during WS more in plants pre-treated with ABA or BA than in those pre-treated with water, but the degree of their de-epoxidation was highest in the later. Similarly, the efficiency of photosystem 2, determined as variable to maximal Chl fluorescence ratio, was not markedly decreased in bean plants pre-treated with ABA or BA in contrast to those pre-treated with water. The imposed WS was not severe enough to damage chloroplast ultrastructure. However, different changes in a size of starch inclusions were observed. In bean plants, the amount of starch increased considerably in plants pre-treated with water, while it decreased in BA pre-treated plants and no change was found in ABA pre-treated ones. The starch content declined under WS in sugar beet and tobacco plants but only moderate changes were found in ABA or BA pre-treated plants. Thus the application of BA and especially of ABA reduced the negative effects of subsequent WS.

Differences in photosynthetic activity, chlorophyll and carotenoid levels, and in chlorophyll fluorescence parameters in green sun and shade leaves of Ginkgo and Fagus

The differences in pigment levels and photosynthetic activity of green sun and shade leaves of ginkgo (Ginkgo biloba L.) and beech (Fagus sylvatica L.) are described. Sun leaves of both tree species possessed higher levels in chlorophylls (Chl) and carotenoids on a leaf area basis, higher values for the ratio Chl a/b and lower values for the ratio Chl/carotenoids (a+b)/(x+c) in comparison to shade leaves. The higher photosynthetic rates P(N) of sun leaves (ginkgo 5.4+/-0.9 and beech 8.5+/-2.1 micromol m(-2)s(-1)) were also reflected by higher values for the Chl fluorescence decrease ratios R(F)(d) 690 and R(F)(d) 735. In contrast, the shade leaves had lower P(N) rates (ginkgo 2.4+/-0.3 and beech 1.8+/-1.2 micromol m(-2)s(-1)). In both tree species the stomatal conductance G(s) was significantly higher in sun (range: 70-19 1 mmol m(-2)s(-1)) as compared to shade leaves (range: 5-55 mmol m(-2)s(-1)). In fact, at saturating light conditions there existed a close correlation between G(s) values and P(N) rates. Differences between sun and shade leaves also existed in several other Chl fluorescence ratios (F(v)/F(m), F(v)/F(o), and the stress adaptation index Ap). The results clearly demonstrate that the fan-shaped gymnosperm ginkgo leaves show the same high and low irradiance adaptation response as the angiosperm beech leaves.

Ontogenetic approach to the assessment of plant resistance to prolonged stress using chlorophyll fluorescence induction method

The review deals with objective reasons that limit the use of chlorophyll fluorescence induction kinetics (Chl FIK) method in plant ecology. Based on the ontogenetic approach (analysis and comparison of the dynamics of the Chl fluorescence ratio Fp/Fs and physiological characteristics of plant leaves in ontogeny) possible criteria for the estimation of general plant resistance of photosynthetic apparatus (PSA) to prolonged stress affects are proposed. One of these criteria is the presence or absence of a steady-state phase in the dynamics of Chl fluorescence ratio Fp/Fs (or Rfd) of plant leaves after they stop growing. We also propose to use the duration of the steady-state phase and variability of Chl fluorescence ratios Rfd and Fp/Fs in this period for quantitative assessment of plant PSA resistance to prolonged stress during plant leaf ontogeny.

Carotenoid composition and photochemical activity of four sandy grassland species

The photosynthetic pigments and photochemical efficiency of photosystem 2 (PS2) were studied in four constitutive species (Achillea millefolium L., Festuca pseudovina Hack. ex Wiesb., Potentilla arenaria Borkh., and Thymus degenianus Lyka) of a semiarid grassland in South-eastern Hungary. Every species displayed typical sun-adapted traits and substantial plasticity in the composition and functioning of the photosynthetic apparatus. The contents of chlorophylls (Chls) and carotenoids (Cars) on a dry matter basis declined from May to July, however, the amount of total Cars on a Chl basis increased. This increase was the largest in Potentilla (48 %) and the smallest in Achillea (14 %). The pool of xanthophylls (VAZ) was between 25 % and 45 % of the total Car content and was larger in July than in May. The content of β-carotene increased by July, but lutein content did not change significantly. The Chl fluorescence ratio Fv/Fm was reduced by 3-10 % at noon, reflecting the down-regulation of PS2 in the period of high irradiance and high temperature. The occurrence of minimal values of ΔF/Fm' showed close correlation to the de-epoxidation rate of violaxanthin. Hence in natural habitats these species developed a considerable capacity to dissipate excess excitation energy in the summer period in their photosynthetic apparatus through the xanthophyll cycle pool and a related photoprotective mechanism, when the photochemical utilization of photon energy was down-regulated.

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R<sub>Fd</sub> of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters Fv, Fm, F0, Fm′, Fv′, NF, and ΔF, the Chl fluorescence ratios Fv/Fm, Fv/F0, ΔF/Fm′, as well as the photochemical (qP) and non-photochemical quenching coefficients (qN, qCN, and NPQ). qN consists of three components (qN = qE + qT + qI), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, qN, qCN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. qN was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas qCN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio RFd (Fd/Fs), which, when measured at saturation irradiance is directly correlated to the net CO2 assimilation rate (P N) of leaves. This RFd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the RFd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.

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, RFd (defined as Chl fluorescence decrease Fd from maximum to steady state fluorescence Fs:Fd/Fs), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio RFd is linearly correlated to the net photosynthetic CO2 fixation rate PN as is indicated here for sun and shade leaves of various trees that considerably differ in their PN. Imaging the RFd-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.

Application of a Chlorophyll Fluorescence Sensor to Detect Chelate-Induced Metal Stress in Zea mays

Metal stress was induced in maize (Zea mays L.) by the addition to the soil of a range of concentrations of either ethylene-diamine-tetra-acetate (EDTA) or citric acid (CA) as chelating agents. Measurements were taken using a recently-developed sensor capable of plant fluorescence detection at wavelengths of 762 and 688 nm. Atmospheric oxygen absorbs radiation at these wavelengths. As such, measured fluorescence can be attributed to the plants under observation. Red/far-red (690/760 nm, R/FR) chlorophyll (Chl) fluorescence ratios were measured before addition of the chelating agents and during the month following. Significant differences were seen in the fluorescence responses of those plants for which high concentrations [≥ 30 mmol kg−1(d.m. soil)] of EDTA were added to the pots compared to those for which CA or no chelating agent was added. The plants for which high concentrations of EDTA were added also exhibited higher tissue metal concentrations and demonstrated visible signs of stress. Before signs of visual stress became apparent, R/FR Chl fluorescence ratios for metal-stressed plants were significantly different to those observed for unstressed plants. These results support the use of plant fluorescence as a potential tool for early indication of phytotoxic metal stress.

The F684/F735 chlorophyll fluorescence ratio: a potential tool for rapid detection and determination of herbicide phytotoxicity in algae

The use of herbicides constitutes the principal method of weed control but the introduction of these compounds into the aquatic environment (primarily through runoff) may have severe consequences for non-target plants. In this study, we describe a sensitive and inexpensive method for detection of photosynthesis-inhibiting herbicides, based on chlorophyll (Chl) fluorescence emission. Algae exhibited a Chl fluorescence signature with two maxima around 684 and 735 nm, correlated with the total Chl content of the algal suspension. The ratio of these two maxima (i.e. F684/F735) can be used as an indicator of stress in the photosynthetic apparatus, and thus represents a very simple method for in vivo evaluation of the health status of algae. Determination of the F684/F735 fluorescence ratio revealed the presence and phytotoxicity of atrazine, metribuzin, terbuthylazine, diuron, DCPMU, DCPU and paraquat. The toxic effect of these pollutants was estimated by monitoring the increase in the F684/F735 value, which reflects photosystem II and photosystem I photochemistry. We observed a drastic increase in the magnitude of this ratio, correlating quantitatively with herbicide concentration and corresponding to a decline in algal photosynthetic activity. For the tested herbicides affecting photosynthetic electron transport, the magnitude of the effect was as follows: diuron=DCPMU>metribuzin>atrazine>terbuthylazine>paraquat>DCPU. The F684/F735 Chl fluorescence ratio thus gives toxicity responses which compare favourably with tests such as the algal growth inhibition test, and could therefore be used to detect the presence and phytotoxicity of herbicides in aquatic environments.

Opposite effects of exogenous sucrose on growth, photosynthesis and carbon metabolism of in vitro plantlets of tomato ( L. esculentum Mill.) grown under two levels of irradiances and CO 2 concentration

Summary The long-term effects of exogenous sucrose (3 percnt;) on growth, photosynthesis and carbon metabolism of in vitro tomato plantlets were investigated under two sets of growth conditions that respectively favor source- or sink-limitations of photosynthesis: 1) low photosynthetic photon flux (PPF) (50 μmol m −2 · s −1 ) and low CO 2 concentration (400 μmol mol −1 ) and 2) high PPF (500 μmol m −2 · s −1 and high CO 2 concentration (4000 μmol mol −1 ). The supply of sucrose under source-limitation conditions increased the growth, the maximal photosynthetic rate, the chl content, the maximal quantum yield of Photosystem II estimated by the Fv/Fm chl fluorescence ratio as well as the soluble sugars (hexoses, sucrose) and starch contents in roots, young and mature leaves when compared to those of photo-autotrophic plantlets. Also, sucrose feeding under these conditions strongly increased the activity of sucrose synthase (SS) (EC 2.4.1.13) in roots and young leaves whereas the activities of sucrose phosphate synthase (SPS) (EC 2.4.1.14), acid invertase (INV) (EC 3.2.1.26) and ADP-glucose pyrophosphorylase (ADPGppase) (EC 2.7.7.27) were highly stimulated in roots and mature leaves. Contrary to these observations, the supply of sucrose to plantlets developed under high PPF and CO 2 concentration decreased growth and led to a somewhat lower maximal photosynthetic rate relative to photo-autotrophic plantlets. These negative responses to exogenous sucrose were accompanied by stronger accumulations of hexose and starch, larger stimulation of INV in mature leaves developed under conditions of sink limitation than those from source limitation conditions. Moreover, under high PPF and high CO 2 concentration, exogenous sucrose led to a marked repression of the SPS activity and caused much lower stimulations of ADPGppase in mature leaves than those observed at low PPF and low CO 2 concentration. We therefore conclude that under our experimental conditions, the interactive effects of exogenous sucrose and environmental conditions on growth and photosynthesis could be rationalized by the source-sink equilibrium of the in vitro tomato plantlets.

Detection of photosynthetic activity and water stressby imaging the red chlorophyll fluorescence

It is shown that with a new flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS), the photosynthetic activity of several thousand points of an intact attached leaf can be screened in a non-destructive way within a few seconds. The method allows the detection of the gradients in photosynthetic capacity over different parts of the leaf. The photosynthetic activity is sensed via imaging the Chl fluorescence at its maximum Fm and at steady state Chl fluorescence Fs of the induction kinetics and by the subsequent determination of the image of the fluorescence decrease ratio (R Fd) which is known as the vitality index of the photosynthetic apparatus. Under water stress the photosynthetic activity decreases as seen in the images of the two fluorescence ratios R Fd and Fm/Fs. Histogram and profile analysis of Chl fluorescence images and Chl fluorescence ratios allow the quantification of the differences between normal and stressed leaves with a high statistical significance.