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Plant Physiology 132:485-493 (2003) © 2003 American Society of Plant Biologists Rapid, Noninvasive Screening for Perturbations of Metabolism and Plant Growth Using Chlorophyll Fluorescence Imaging1Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom (R.P.B., K.O., N.R.B.); and Bayer CropScience GmbH, Ecotoxicology, Industriepark Höchst, 65926 Frankfurt am Main, Germany (K.E.P.)
A rapid, noninvasive technique involving imaging of chlorophyll fluorescence parameters for detecting perturbations of leaf metabolism and growth in seedlings is described. Arabidopsis seedlings were grown in 96-well microtitre plates for 4 d and then treated with eight herbicides with differing modes of action to induce perturbations in a range of different metabolic processes. Imaging of chlorophyll fluorescence emissions from 96 seedlings growing on a microtitre plate enabled images of a number of fluorescence parameters to be rapidly and simultaneously produced for the plants in each well. Herbicideinduced perturbations in metabolism, even in metabolic reactions not directly associated with photosynthetic metabolism, were detected from the changes in the images of fluorescence parameters considerably before any visual effects on seedling growth were observed. Evaluations of seedling growth were made from measurements of the area of chlorophyll fluorescence emission in images of plants growing in the 96-well plates. Decreased seedling growth related directly to herbicideinduced changes in the imaged chlorophyll fluorescence parameters. The applicability of this rapid-screening technique for metabolic perturbations in monocotyledonous species was demonstrated by treating Agrostis tenuis seedlings with Imazapyr, an inhibitor of branched-chain amino acid synthesis.
In many areas of plant biology and agrochemical research, there is an increasing requirement for more rapid-screening techniques to identify plants with impaired metabolism and growth. The rapid expansion of Arabidopsis genomics research programs has highlighted the need for new technologies to facilitate the identification of potentially interesting mutants with genetic modifications that impair specific metabolic reactions and growth (e.g. Borevitz et al., 2000  ; Nakazawa et al., 2001). Also, within the agrochemical industry, more efficient screening processes for crop protection chemical discovery are needed to deal effectively with the increases in the numbers of molecules entering the initial cascade of screening. For many years, conventional screening methods involved screening 5,000 to 20,000 new chemical entities per year in greenhouse screens, which for herbicides involved pot-grown plants in greenhouses (Evans, 1999). Combinatorial chemistry can provide at least a 10-fold increase in the numbers of molecules for screening, however, quantities of each molecule are normally less than 1 to 2 mg, which is insufficient for conventional greenhouse screens. In parallel to the introduction of combinatorial chemistry has been the introduction of new microscreening or high-throughput screening, which, for herbicide screening, involves growing plants in 96-well microtitre plates (Berg et al., 1999; Evans, 1999). In the past, conventional greenhouse screening has involved detailed visual assessment of herbicidal activity over a 2- to 3-week period following treatment. This is not possible using microscreening or high-throughput screening, and less time-consuming and automated assessment procedures are required (Ridley et al., 1998).
It has long been known that chlorophyll fluorescence emission kinetics from plants provide an indicator of plant photosynthetic performance (McAllister and Myers, 1940 In this study, we demonstrate how chlorophyll fluorescence imaging can be effectively and rapidly used to identify perturbations of leaf metabolism considerably before the onset of any visual effects on leaf morphology or plant growth. A range of herbicides with differing modes of action was used to perturb leaf metabolism. These have clearly defined molecular targets and were chosen to reflect interference with a range of essential metabolic processes. An analysis is presented of the most appropriate fluorescence induction parameters to use for the rapid detection of such metabolic perturbations and the associated small changes in growth.
Chlorophyll Fluorescence Parameters The protocol for taking images of chlorophyll fluorescence and determining fluorescence parameters is shown in Figure 1. When untreated Arabidopsis plants that have been kept in the dark for 15 min are exposed to the weak measuring beam of the fluorescence imaging instrument, chlorophyll fluorescence emission from the plants rises to the minimal level of fluorescence (Fo), which is the fluorescence level obtained when the photosystem II (PSII) centers are in the "open" state (capable of photochemistry). Exposing plants to a saturating light pulse with a photosynthetic photon flux density (PPFD) of approximately 4,000 µmol m–2 s–1 for 0.8 s drives a very high proportion of the PSII centers into the "closed" state (making the capacity for photochemistry close to zero) and results in the maximal level of fluorescence (Fm). After termination of the saturating flash, the fluorescence rapidly falls to a steady state and the plants are then exposed to an actinic PPFD of 30 µmol m–2 s–1, which generates rapidly the transient fluorescence level, Fi, before a peak value of fluorescence, Fp, is reached (Fig. 1). The rapid rise to Fi reflects an increase in the yield of charge stabilization at PSII that is independent of subsequent changes in the redox state of the plastoquinone pool. Changes in the fluorescence level between Fi and Fp are almost entirely due to increased reduction of the plastoquinone pool, which is largely determined by the relative rates of charge stabilization at PSII and plastoquinol oxidation at the cytochrome b6f complex.
It has previously been shown that perturbation of leaf metabolism by a wide range of herbicides with different modes of action will modify the fluorescence induction characteristics of leaves (Blowers, 1989
The effects of the application of a range of concentrations (0.4–8 mM) of Imazapyr to 4-d-old Arabidopsis seedlings after 24 and 48 h are shown in Figure 2. After 24 h of treatment, there are no marked visual effects on plant growth and development (Fig. 2, A and B), however marked changes in fluorescence characteristics were observed (Fig. 2, D and E). Images of the fluorescence parameter Fv/Fm, which has been widely used to detect stress in plants (Maxwell and Johnson, 2000
It has been previously shown that herbicideinduced modifications to fluorescence induction kinetics can result in modifications not only to Fv/Fm, but also to the parameters Fo/Fp, Fo/Fi, and Fi/Fp (Habash et al., 1985
The sensitivity of the fluorescence screen can be improved by increasing the numbers of replicates being imaged. Using eight replicates for the treatment of plants with 0.8 mM Imazapyr (see Fig. 2) the decrease in Fv/Fm only becomes significant after 24 h (Fig. 3A). When the number of replicates imaged was increased to 16, a significant decrease in Fv/Fm at P < 0.05 was observed after only 6 h of treatment (Fig. 4). Increasing the number of replicates to 32 and 64 did not result in an improvement of the P values for significant difference (Fig. 4).
An important potential use of these fluorescence screens will be for the detection of metabolic perturbations in mutagenized plant populations. The very high sensitivity of the screening technique may often result in the identification of very many small metabolic perturbations, which may not be of major interest and on which, due to the large numbers, it would be practically difficult to conduct detailed metabolic analyses. In such cases, it is important to set appropriate selection thresholds for percentage change in the fluorescence parameter to ensure sensible numbers of plants are selected for further time-consuming, detailed analyses that can determine the mechanistic bases of the perturbations.
To examine the suitability of the fluorescence screen for identifying a range of metabolic perturbations, Arabidopsis seedlings were treated with seven other herbicides (Asulam, Atrazine, Bifenox, 2,4-dichlorophenoxyacetic acid [2,4-D], Diclofop-methyl, Glyphosate, and Paraquat) with differing modes of action (see Table I), and the fluorescence parameters were imaged. The changes in Fv/Fm, 1–(Fo/Fp), 1–(Fo/Fi), and 1–(Fi/Fp) 6 and 48 h after spraying the seedlings with these herbicides at their recommended field application rates are shown in Figure 5. After 6 h, significant decreases in all of the fluorescence parameters could be detected for plants treated with Asulam, Atrazine, Diclofop-methyl, Glyphosate, and Paraquat, whereas the changes induced by Bifenox and 2,4-D were not significant. After 48 h, significant decreases in Fv/Fm, 1–(Fo/Fp), and 1–(Fi/Fp) were observed with all of the herbicides, although no significant decreases were observed in 1–(Fo/Fi) of plants treated with Atrazine, 2,4-D, and Bifenox. Clearly, the fluorescence parameters Fv/Fm, 1–(Fo/Fp), and 1–(Fi/Fp) can be used successfully to detect a number of very different metabolic perturbations in leaves.
Although there is a considerable need to develop improved screening techniques for metabolic deficiencies in Arabidopsis, this is not a good model for studying metabolic perturbations in monocotyledonous species. A number of herbicides specifically target monocotyledonous species by exploiting metabolic differences between monocotyledonous and dicotyledonous plants (Devine and Preston, 2000
Previously, experiments with a range of other species (Alopecurus myosuroides, Avena fatua, bean [Phaseolus vulgaris], white mustard [Sinapis alba], wheat [Triticum aestivum], and maize [Zea mays]) have also demonstrated the wide applicability of these fluorescence parameters for the detection herbicideinduced perturbations of metabolism (Habash et al., 1985
The software of the chlorophyll fluorescence imaging system allows determination of the area within each well of the microtitre plate that generates a fluorescence signal. The calculation of this area is independent of chlorophyll concentration and fluorescence yield. Consequently, provided chlorophyll is distributed across the whole of the leaf, i.e. there are no achlorophyllous regions as found in variegated leaves, leaf area can be estimated accurately from the area of chlorophyll fluorescence emission at the Fm level taken from images. Measurements of the fluorescent leaf area can be used to give a rapid and accurate indication of plant growth at early stages of plant development when it can be difficult to detect differences in growth by eye. In Arabidopsis, the decreases in the fluorescence parameters induced by treatment with Imazapyr (Figs. 2 and 3) would be expected to result in decreases in photosynthetic productivity and growth. The relationship between the decreases in Fv/Fm and the fluorescent area for plants treated with 0.4, 0.8, and 4 mM Imazapyr for 6 and 48 h is shown in Figure 7. A strong correlation exists between Fv/Fm and the area of leaves exhibiting chlorophyll fluorescence, thus demonstrating that Fv/Fm is a good indicator of plant growth in these experiments. A similar close correlation between 1–(Fi/Fp) and leaf area was also observed (data not shown), confirming that this fluorescence parameter could also be used as an effective screen for metabolic perturbation of plant growth.
The area emitting fluorescence from the leaves of Arabidopsis seedlings growing in 96-well microtitre plates is a good indicator of plant growth because the leaves are planophile with little overlap at early growth stages. However, when there is considerable overlap of leaves or for non-planophile leaf growth, as shown by monocotyledonous plants such as A. tenuis, this may not be always the case. No clear relationship was found in A. tenuis leaves treated with Imazapyr between Fv/Fm and the area of leaves exhibiting chlorophyll fluorescence (data not shown), presumably due to the considerable overlapping of the leaves preventing accurate determinations of area. Careful examination of the relationship between fluorescent leaf area and growth should be made for any experimental system before routinely using fluorescent area to screen for growth differences.
This study has demonstrated the effectiveness of chlorophyll fluorescence imaging for rapidly detecting perturbations in leaf metabolism before any effects on growth and development are visually detected. An important advantage of fluorescence imaging over integrated measurements of fluorescence using conventional fluorimeters for screening of metabolic perturbations is the ability to monitor simultaneously 96 seedlings growing in a 96-well microtitre plate. Larger plants can be also be screened in larger well-plate formats, but increasing plant size will obviously decrease the number of plants that can be screened simultaneously. It should be stressed that not all metabolic perturbations will be detected by fluorescence imaging, but a surprisingly large proportion will be. This screen will not detect alterations in metabolism that do not impact directly or indirectly on photosynthetic metabolism. However, many perturbations of metabolic reactions that would not be predicted to be linked in any way with photosynthetic metabolism have been found to modify chlorophyll fluorescence. Chlorophyll fluorescence imaging can also be used for rapidly estimating differences in growth of seedlings with planophile, nonoverlapping leaves. The total area from which fluorescence at Fm is emitted from each plant is directly related to leaf area that contains chlorophyll. For seedlings with nonplanophile growth habit or which have large areas of leaf overlap, the correlation between the fluorescent leaf area and plant growth can be poor and should be examined carefully before using the fluorescencent area to screen for growth differences.
Plant Material
Seeds of Arabidopsis were soaked in sterilized water containing 0.1% (v/v) Tween 20 for 30 min and then sterilized by adding an equal volume of commercial bleach containing approximately 7% (w/v) sodium hypochlorite and shaking for 90 s. The seeds were then washed six times in sterilized water, kept in the dark at 4°C for 4 d and sown into sterilized 0.8% (w/v) agar containing Murashige and Skoog basal medium (Murashige and Skoog, 1962 Agrostis tenuis plants were similarly grown from seed except that the growth medium was sterilized 0.8% (w/v) agar and eight seeds were sown in each well. Plants were treated with herbicides 4 d after sowing.
A range of herbicides with differing modes of action were applied to leaves as a spray at molar concentrations determined from their average field use rates (grams or kilograms per hectare) given by Tomlin (2000
Images of chlorophyll fluorescence parameters were obtained using a FluorImager chlorophyll fluorescence imaging system (Technologica Ltd., Colchester, UK). All lighting (actinic, saturating pulses for measurement of Fm and Fm' and the measuring pulses applied while imaging) is provided from 16 banks of 100 blue light-emitting diodes (peak output 470 nm). The required incident PPFD at any point is generated through pulse-width modulation at constant voltage, which maintains a constant spectral output. Images are taken using an asynchronous progressive scan CCD camera, which is synchronized to the measuring pulses from the light emitting diodes. Fo was measured during the weak measuring pulses. Fm was measured during an 800-ms exposure to a PPFD of approximately 4,000 µmol m–2 s–1. During the last 650 ms, the camera took a train of images at 20 Hz (2-ms exposures at low camera gain). The program then searches these images for the one with the highest mean value. This image is saved and is used in construction of the Fv/Fm image. For a usable fluorescence image to be generated, the CCD must absorb a minimum number of photons. Increasing the exposure time and/or incident, PPFD will increase the number of photons accumulated. However, when imaging chlorophyll a fluorescence, increasing either of these will often impact on the de-excitation processes at PSII, a problem that is most acute when imaging Fo. Also, exposure time is often limited by the accumulation of long wavelength photons (dark noise). With the FluorImager system, an Fo image is generated within a 16.7-ms exposure, during which 2-µs pulses (PPFD of 4,000 µmol m–2 s–1) are applied every 300 µs. Although the same number of photons could be delivered during a single pulse of approximately 110 µs at the same PPFD, the long exposure time at low average PPFD has the advantage of allowing for the opening of PSII centers, thereby increasing the accuracy of Fo measurement.
Fi is the inflection in fluorescence level that occurs during the rapid rise in fluorescence when a dark-adapted leaf is exposed to actinic light (Fig. 1; Habash et al., 1985 Although up to 16 fluorescence traces can be generated from different areas of the image simultaneously, only one complete trace (which represented a mean of the entire well plate) was routinely generated during these experiments. The FluorImager system allows for images to be divided into 96 zones (one for each well of the micotitre plate in which Arabidopsis plants are grown) through the application of horizontal and vertical lines. The program automatically calculates mean values from each zone and also determines the active (fluorescent) area within each zone. Fluorescence induction curves are also generated for each zone, and images of 1–(Fo/Fp), 1–(Fo/Fi), and 1–(Fi/Fp) were constructed. All nonimage data (induction curves from each zone and fluorescence parameter values) were copied and pasted into Excel (Microsoft Corporation, Redmond, WA) for analysis. The FluorImager software allows determination of the area that has generating a fluorescence signal in any given image. Calculation of this area is independent of chlorophyll concentration and fluorescence yield. For Arabidopsis plants, the area of leaf in each well was estimated from the area of the fluorescence emission at the Fm level and was used to monitor plant growth. Received November 22, 2002; returned for revision January 7, 2003; accepted February 8, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.018093.
1 This work was supported by the European Commission under the Marie Curie Industrial Host Fellowship Program (grant no. HPMI–CT–1999–00085 to Bayer CropScience GmbH). * Corresponding author; e-mail baken{at}essex.ac.uk; fax 44–1206–873416.
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ABSTRACT
RESULTS AND DISCUSSION
) and for leaves treated with 4 (
) and 8 (
) mM Imazapyr for 24 h.
), and 50% (v/v) acetone containing 0.1% (v/v) Tween (
). Data are the means of eight replicates; SEs are shown when larger than the symbol.
