L. Chaerle 1 , R. Valcke 2 , and D. Van Der Straeten 1,*
1 Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35,
B-9000 Ghent, Belgium.
2 Limburgs Universitair Centrum, Universitaire Campus, Dept. SBG, B-3590
Diepenbeek, Belgium
* Corresponding author e-mail: dostr@gengenp.rug.ac.be; Fax: +32 9 264 5333
1. INTRODUCTION
STRESS MONITORING IN PLANTS - SCOPE OF THE PRESENT REVIEW
By using imaging techniques, plant physiological parameters can be assessed in vivo without contact with the plant and in a non-destructive way, from a microscopic to a remote sensing scale. The major advantage of imaging consists in the instantaneous visualisation of heterogeneity in the studied parameter of a leaf or leaf area. To obtain such a detailed overview, classical techniques would need a multitude of repeated point measurements at each time point. Moreover, measurement on the same minute regions of a sample at each measuring session is nearly impossible. Especially destructive measurements are labour intensive and time consuming, and one has to assume that the different samples used for each determination are physiologically
'comparable'. Thus imaging techniques are an ideal method to visualise the evolution of patterns of plant physiological parameters.
Imaging has a vast potential for highlighting local changes in a parameter from surrounding 'background' levels. This discriminating power of imaging techniques allows an early detection of deviations from optimal physiological conditions. Imaging in the visual spectrum is a popular technique to record long-term physiological processes as growth and development. This type of monitoring is frequently designated time-lapse imaging. However, in addition to visual assessment, imaging in other spectral regions permits to reveal other fundamental plant physiological parameters as transpiration and chlorophyll fluorescence. Imaging in narrow spectral bands, coined hyperspectral imaging, makes it possible to extract information regarding concentrations of specific compounds in plants. Microscopic techniques, which will be mentioned briefly in this review, are generally applied for fundamental research on cellular and subcellular changes during development or upon stress. This still holds for the macroscopic scale, covering measurements on plant organs or single plants.
At canopy and field scale, imaging techniques have the potential to identify emerging stresses and to guide sampling for identification of the stressor. These remote sensing applications are increasingly being applied in the rapidly evolving precision agriculture, which should improve crop production, while being more environmentally friendly than the 'classic' production methods

2. THERMOGRAPHY
At ambient temperature, all objects emit far infrared light of approximately 10 μm wavelength (Nobel, 1991). Detectors sensitive in the 814 μm wavelength band convert this radiation into a temperature reading. Such detectors are the basis of nonimaging infrared thermometers, which yield an average temperature measurement of all objects within the field of view. Applications of these simple and affordable instruments include forest canopy studies and irrigation scheduling in field crops (Samson and Lemeur, 2000; Wanjura and Upchurch, 2000). Images can be generated from a thermal infrared detector by adding a scanning system. Each point measurement is assigned a pseudocolour value depending on the radiation captured. In this way patterns of radiation are converted to visual pseudocolour images representing temperature levels. Scanning thermal imaging systems have been used to monitor temperature distribution at the plant level since the mid-seventies (De Carolis et al., 1975).
The last few years a new generation of thermal imaging systems has been commercialised. The use of arrays of detectors obviates the incorporation of expensive highspeed scanning systems. Moreover sensors that can operate at ambient temperature have been developed, whereas previously detectors needed to be cooled cryogenically to obtain a temperature resolution of 0 .1 °C. These technical improvements make thermal cameras more affordable and user friendly, promoting an increase in industrial and expectedly in biological applications (Majumdar and Norton, 1999)
The energy budget of a plant, and thus its temperature is dependent on environmental factors. Sufficient light and water are the main factors in the energy budget and a prerequisite for satisfying yield. The most common plant stress is water shortage, which results in a higher leaf temperature due to decreased transpirational cooling. Most applications of thermography in plant physiology are based on monitoring changes in transpiration.
2.1. Monitoring of thermogenic flowering
As an exception in the plant world, flowers from a few plant families have the capacity to generate heat, which is used to attract pollinators by volatilising attractants.
Thermography has been used to study the timing of this thermogenesis phenomenon
(Raskin et al., 1989). On the other hand, metabolism has a negligible influence on plant leaf temperature. Heat can be accumulated only in tissues that have a high heat capacity, such as the specialised flower structures mentioned above, (Breidenbach et al., 1997).
2.2. Thermographic visualisation of freezing
While evaporation uses latent heat, and thus has a cooling effect on leaves, freezing processes liberate latent heat. Thermal imaging was successfully applied to detect locally initiated freezing processes in plants (Pearce, 2001). It is particularly important to correlate the speed and spread of freezing with subsequent visible freezing damage (Pearce and Fuller, 2001). Such thermal studies of freezing processes in function of time can aid in developing frost-protection strategies (Wisniewski et al., 1997).
2.3. Thermography in mutant screening

In case of water shortage, plants limit transpiration by closing their stomata. Stomata can be seen as microscopic valves that optimise the uptake (and thus assimilation) of
CO
2
while limiting the loss of water. Under water stress, stomata close via a process controlled by abscisic acid (ABA) (Hetherington, 1998). Since thermography readily visualises changes in transpirational cooling, this imaging technique is an efficient tool to screen plant populations for aberrant transpiration. Initially, thermography was used to isolate ABA-insensitive mutants in barley (Raskin and Ladyman, 1988). In recent years the technique has been applied to screening of the model plant Arabidopsis (Figure 1), since hormonal regulation of plant transpiration is a logical target for thermographic screening (Merlot et al., 2001). Thermal imaging is also well suited to check stomatal functionality in mutants isolated with other screening methods
(Gray et al., 2000).
Figure 1. Thermal imaging of drought stressed Arabidopsis .
Control plants (top row), an ABAinsensitive mutant ( abi-2-1 , middle row) and an abi-2-1 revertant showing a suppression of ABA-insensitivity
( abi2-1R1 , bottom row) were 17 days old when subjected to four days of drought stress. Leaf temperature of
ABA-insensitive plants was 1 to 1.5
°C lower when compared to the control plants, whereas the temperature of abi2-1R1 plants was comparable to that of wild-type. The abi2-1R1 mutant was isolated during a thermographic screen of a drought-stressed mutagenised abi-2-1 population for plants with a higher leaf temperature. The scale is in °C. (reproduced with permission from Merlot et al., 2001.)
2.4. Estimation of transpiration
Although several physiological methods are available to measure transpiration, none of them can be easily applied at the field scale, neither do they permit long-term continuous measurements on a growing crop (Merta et al., 2001). By virtue of its noncontact methodology, thermography can be applied at any scale from the laboratory
(Figure 2) to air-borne field applications. Moreover, automatic surveillance of the water status of a crop is an application within reach of current technological possibilities
(Jones, 1999a). Since plant leaf temperature is dependent on weather conditions, the application of thermography to assess water stress on the field is proposed in conjunction with a dedicated calibration method, to compensate for the variable environmental conditions (Jones, 1999b). Importantly, stomatal closure also affects photosynthesis (Osmond et al, 1998; Cornic, 2000; Baker et al., 2001). Thus thermal imaging could complement chlorophyll fluorescence imaging measurements, making it possible to discern water stress from other stresses affecting chlorophyll fluorescence. This could be achieved with a combined portable thermal and fluorescence
(multispectral) monitoring system (see 5.2).

2.5. Detection of pathogen infections
Figure 2. Thermographic visualisation of the ‘Iwanov’ effect in the first trifoliate leaf of
French bean. The leaf was severed from the plant 10s after the first image was taken. The response results from cutting the leaf from the plant and consequently interrupting its water supply. The image series was captured with 1-minute intervals. First, a rapid cooling is apparent, characterised by initial stomatal opening. The onset of rewarming (see temperature indications in the images of the last row) is due to stomatal closure as the leaf water status declines. These changes in stomatal aperture, and thus transpiration and evaporative cooling, cause a decrease in leaf temperature followed by an increase. The temperature indications on each figure indicate the average temperature of the same 1cm 2 region on the leaf surface (Reproduced with permission from Jones, 1999b).

During plant-pathogen infection, the physiological state of the invaded tissue is altered. This can be reflected in changes in photosynthesis, transpiration or both. Fluorescence imaging (see 5.2) and thermography are thus useful for rapid visualisation of emerging biotic stresses.
Some compounds produced by pathogens are known to induce stomata closure.
Examples are hydrogen peroxide (McAinsh et al., 1996), elicitors generated during infection of tomato (Lee et al., 1999), toxins produced by the plant-pathogenic bacteria Pseudomonas syringae (Mott and Takemoto, 1989) and an unidentified product produced during the infection of soybean by the phytopathogenic fungus Phytophthora (McDonald and Cahill, 1999). Salicylic acid (SA), the central signalling component in the disease resistance response of many plant species (Delaney et al., 1994), also diminishes stomatal aperture (Larqué-Saavedra, 1979; Manthe et al., 1992). The stomatal movements that occur during plant-pathogen interactions can be imaged by thermography. A few examples are described below.
In the well-characterised plant-pathogen model system tobacco - tobacco mosaic virus (TMV), resistance is accompanied by a necrosis response termed hypersensitive response (HR). In this incompatible plant-pathogen interaction, SA starts to accumulate before the appearance of visible necrosis (Enyedi et al., 1992; Malamy et al., 1992). As was hypothesised from the above facts, thermography monitored an early increase in leaf temperature at the points of TMV-infection (Chaerle et al., 1999,
Figure 3). The visualisation of the emerging 'hot-spots' was presymptomatic, because the first signs of necrosis only became visible 8 hours later. Moreover, the full extent of the thermal effect corresponded with the area of cell death 4 days later. The concentration of water vapour in the air circulated through a cuvette clamped onto an infected leaf area, as measured by continuous infrared gas analysis (IRGA), revealed a local decrease in transpiration. This decrease correlated with the increase in leaf temperature measured thermographically. A robotised system coupled to an 'on-line'
'real-time' continuous visualisation method (see www.plantgenetics.rug.ac.be/~lacha) was used for thermography-aided selective sampling of visually asymptomatic tissue.
The increase in SA content of these samples as a function of time corresponded well with the measured decrease in transpiration as infection progressed. Such site-
Figure 3. Two pairs of infrared and visual spectrum images at an early and a late stage of the incompatible interaction between tobacco and tobacco mosaic virus
(TMV). The plant was infected and kept at 32°C for 29 hours to allow the spread of
TMV. The plant was then shifted back to 21°C. Two and a half hours after the temperature shift a thermal effect emerged. The upper left panel shows the presymptomatic thermographic visualisation of the infection 8 hours after the temperature shift.
The right panel shows the video image taken at the same time. No signs of cell death are yet visible. The lower left and right panels were captured 122 hours post temperature shift, and show extensive cell death at the places of infection (Chaerle and Van
Der Straeten, unpublished).

specific sampling could also be applied to the molecular characterisation of the early stages of plant-pathogen interactions. Panoramic views of gene expression during biotic stress were recently obtained using microarrays of a selected set of genes
(Maleck et al., 2000; Reymond, 2001; Schenk et al., 2000). This approach could thus be extended to early symptomless time points by taking advantage of imaging-aided selective tissue sampling.
The agriculturally more important Brassica napus infection with Phoma lingam was also visualised presymptomatically by thermal imaging. The incompatible interaction between Brassica and Phoma results in stem canker, a disease characterised by stem necrosis (Lamkadmi et al., 2000). In addition to the thermal data, changes in protein patterns were detected before the appearance of disease symptoms, and amongst the differentially induced polypeptides, a phosphatase was identified
(Lamkadmi et al., 1996).
Cell death is a common symptom during the HR in incompatible plant-pathogen interactions. In mutants and in some transgenic lines from several plant species, spontaneous cell death occurs (Dietrich et al., 1994; Johal et al., 1995; Mittler and
Rizhsky, 2000). Comparable to the visualisation in the tobacco-TMV system, the onset of cell death could be revealed before the appearance of visual symptoms in lsd 2
Arabidopsis mutants and in tobacco bacterio-opsin transgenics (Chaerle et al., 2001).
Thermography visualised the dynamic evolution and spreading of these cell death phenomena in function of time. Since cell death at the surface of plant leaves results in evaporation of the contents of disrupted epidermal cells, high-contrast images can be thermally recorded.
2.6. Non-destructive testing in biology
Together with process monitoring and preventive maintenance control, nondestructive testing is a frequent application of thermography in many industrial sectors. In the sector of processing of biological materials, thermography can for instance be applied for non-destructive testing in the wood industry (Wyckhuyse and
Maldague, 2001).
Active thermography, which uses pulses from high-power infrared lamps to heat transiently the monitored samples, has also found widespread application in industrial manufacturing. Transient heating permits thermographic detection of local differences in heat capacity, linked to structure or composition, by monitoring the speed of either warming or subsequent cooling. This technique has also been used in medical applications (Fujimasa, 1998) and in post-harvest processing of fruit (Offermann et al., 1998). An agricultural robotics application was developed based on active thermal imaging to detect diseased potato tubers (Lefebvre et al., 1993). Furthermore, the active thermography methodology was used to visualise water distribution in plant leaves (Kümmerlen et al., 1999). This setup monitors the temporal evolution of water content in intact plants, which is impossible with the commonly used destructive methods.
2.7. Thermal remote sensing
Applications at the remote sensing scale were attempted as soon as portable thermal imagers became available, and are presently refined for integration into precision farming (Liu et al., 2000). These measurements however depend on environmental conditions, which influence the thermal properties of the visualised crop. Calibration of images according to weather conditions is necessary for comparison between image data obtained during different measuring periods and growth seasons (Nilsson,
1995). A field trial with winter wheat proved that root nematode infection increases canopy temperature in comparison with a control field (Nicolas et al., 1991). In the case of an economically important rice disease, rice blast, leaf temperature was

shown to correlate with the severity of disease in the field, whereas remote sensing in the visual spectrum could not discern infested from control fields (Yamamoto et al.,
1995).
2.8. In-vitro applications
Robotised thermography is exploited to screen animal cell cultures for their thermal metabolic reaction to pharmacological compounds (Paulik et al., 1998). Such a highthroughput non-destructive testing setup could be used for plant cell cultures as well.
Recently, thermal imaging was applied to monitor in-vitro reactions of enzymes with stereospecific substrates (Reetz et al., 2001). Exothermic reactions show up as hotspots in microtiter plates used for screening. This application is proposed for identification of enantioselective enzymes, which have a broad application range in microbiology and biotechnology.
3. BIOLUMINESCENCE IMAGING
A wide variety of life forms, including insects, jellyfish and bacteria, can emit visible light through enzymatic reactions on high-energy substrates. Substrate availability is the major limitation for the long-term application of these reporter systems in plants.
To detect the relatively weak light emission, measurements have to be carried out in darkness with cooled image-intensified CCD cameras. This technique is essentially different from fluorescence imaging (see 5) where chromophores, as for example
GFP, absorb light from an intense light source and emit light at a lower wavelength.
3.1. Aequorin imaging
The photoprotein aequorin, naturally present in the jellyfish Aequorea victoria , emits blue light upon exposure to free calcium. This Ca 2+ reporter protein consists of a polypeptide apoaequorin and its luminophore coelenterazine. To avoid the invasive method of loading of the calcium reporter molecule aequorin into plant cells, transgenic plants expressing apo-aequorin were generated. Thus, measurements of free calcium concentration dynamics at the whole plant level were made possible (Knight et al., 1991). Aequorin needs to be reconstituted in vivo in apoaequorin- producing transgenic plants by floating seedlings on an aqueous solution of coelenterazine. To localise precisely the emission of aequorin bioluminescence, luminescence images are superimposed with reflection images taken with the same microscope, under dark-field visual spectrum illumination. Since Ca 2+ is a ubiquitous second messenger involved in multiple stress responses, aequorin imaging has been adopted to assess calcium signalling in diverse stress responses, including the oxidative burst after pathogen attack and guard cell signalling (Grant et al., 2000; Wood et al., 2000).
3.2. Luciferase imaging
The luciferase enzyme (LUC), encoded by the firefly luc gene, is a popular in vivo reporter of gene expression. As is also the case for the above-described aequorin imaging, the substrate (luciferin for luciferase imaging) has to be kept at a sufficient concentration either by regular spraying or overnight imbibition. Since luciferase has a half-life of 3 hours, continuous assessment of changes in its expression is possible.
The prime application has been the study of biological rhythms in plants (McWatters et al., 2000; Somers et al., 2000). In addition, plant-pathogen interactions, oxidative stress and wounding have been studied using this reporter system (reviewed in
Chaerle and Van Der Straeten, 2001). Recently, luciferase imaging has been used to isolate mutants in gibberellic acid response (Meier et al., 2001). To spatially correlate the effects of photooxidative stress - as observed with high resolution fluorescence

imaging (see 5.2) - with gene expression, plants were transformed with the LUC reporter under the control of a light-stress induced peroxidase promoter (Baker et al.,
2001). Parallel imaging of chlorophyll fluorescence and luminescence will help to elucidate the effects of light-inhibition of photosynthesis.
A different type of application makes use of the luciferase operon, which encodes both a luciferase and its substrate, present in certain marine bacteria. Plantpathogenic bacteria transformed with this operon can be visualised in planta. This possibility was exploited to selectively sample symptomless tissue already invaded by the pathogen (Gay and Tuzun, 2000). Comparison between the responses of different cultivars to bacteria can help to characterise resistance factors.
4. REFLECTANCE IMAGING
The human vision system is based on detection of reflected radiation in the 400-
800nm spectral range. Consumer photographic emulsions mimic this spectral sensitivity. The spectral sensitivities of custom emulsions and of CCD detectors in video cameras extend into the near infrared. Consumer video cameras have a filter that leaves out the near infrared radiation (NIR). Imaging in the visible spectrum is used to obtain reference images for fluorescence, thermal and bioluminescence imaging.
This approach makes it straightforward to correlate patterns observed in other spectral regions with concurrent or later emerging visual symptoms.
On the microscopic scale, custom-designed measuring cuvettes allow to keep live samples in controlled conditions for long periods of time. Cytological and morphological changes in symbionts and host cells were visualised by time-lapse imaging (Giovannetti et al., 2000). Confocal fluorescence microscopy (see 5.1) of the interaction between Nicotiana roots and arbuscular mycorrhizae (Vierheilig et al., 2001) could be extended in time by using dedicated observation chambers. The same holds for plant-pathogen interactions (Howard, 2001). At the same measuring scale, stomatal movements in response to stress can be imaged in function of time, in a custom-built gas-exchange chamber (Kaiser and Kappen, 2000). Individual stomata within the enclosed 25x50mm leaf area can be imaged repeatedly through electronic remote control. Simultaneous information on stomatal aperture, CO
2 assimilation and transpiration, the latter both being determined on-line using IRGA (see 2.5), provides all necessary information to study the limitation of decreased stomatal aperture on photosynthesis in function of light and/or humidity (see 2.4).
Time-lapse imaging and continuous video-capture have been extensively applied for long-term monitoring of plant growth. To study growth responses in the absence of light, near infrared illumination, which is generally regarded as physiologically inactive, is used. Near infrared radiation (NIR) illumination and imaging under natural light conditions visualises plant leaves with higher contrast compared to visual spectrum imaging. This fact has been exploited to calculate the local speed of leaf growth from time sequences of near infrared images (Schmundt et al., 1998). The resulting data are graphically represented as a displacement map. This a pseudocolour representation of the leaf, where light colours indicate high growth rate, and darker colours slower growth rate The technique was subsequently applied to monitor the effect of drought stress on diurnal variations in leaf growth rate (Schurr et al., 2000). Under field conditions, NIR permits to discern leaf cover from soil (Marchant et al., 1998), what could be applied in multispectral systems to guide leaf-cover-specific extraction of information in other spectral bands with low soil-crop contrast (see 7).
To extend the range of imaging applications, systems can be mounted on autonomous vehicles for field applications or on robotic installations in laboratory and nursery environments. In addition, multiple cameras can be monitored from a central computer system. Multi-site monitoring of the activity of predators on plants infested with insect pests was achieved by the use of 16 multiplexed videocameras equipped with IR-illumination to allow night time measurements as well (Meyhofer, 2001). On

the field scale, simple optical visual spectrum observation was used to quantify herbicide phytotoxicity with vehicle-mounted optical sensing (Bell et al., 2000). This technique was found to be as reliable as conventional visual assessment by an experienced person. Remote sensing techniques are well suited for site-specific crop management and yield prediction, since from the combination of image sets taken at different growth stages of the crop, both spatial and temporal information can be extracted (Shanahan et al., 2001). In the classical 'manual' approach to investigate growth of field crops, repetitive and intensive measurements representative for the whole crop, need to be carried out in the field at regular intervals. Remote sensing provides an accurate alternative on which crop management decisions can be based
(Flowers et al., 2001). Importantly, field measurements should be conducted according to standardised protocols to avoid the influence of sun zenith angle (Dymond et al., 2001) and leaf wetness (Guan and Nutter, 2001). Laboratory measurements are less prone to such influences as a result of controlled environmental conditions.
5. FLUORESCENCE IMAGING
Plants contain compounds that fluoresce upon excitation with short-wavelength light.
Upon excitation with UV or blue light, light of longer wavelength is emitted. The choice of an appropriate filter set on the light source and on the detector allows the light emitted from the target fluorochrome present in the leaf to be imaged only. In addition, the introduction of artificial fluorochromes by loading into cells or by transformation of the plant permits to visualise a whole array of physiological changes.
Endogenous and artificial fluorophores can be excited by illumination and visualised microscopically at the cellular level. On the macroscopic scale, autofluorescence or fluorescence from expressed fluorophores can be captured from plant organs, whole plants or canopies. Possible applications of fluorescence imaging range from analysis of photosynthesis and metabolism to cell biology, gene expression, signalling research and studies of plant-microbe interactions.
5.1. Fluorescence microscopy
By using specific fluorescence probes, the dynamics of cellular substances can be visualised. Fluorescence ion imaging permits to monitor the transport of ions, a mechanism implicated in a multitude of signalling processes in plants (Roos, 2000).
Microscopic observation of cellular processes has been greatly improved by techniques to express fluorochromes in plants under the control of specific promoters, or as fusion proteins. The green fluorescent protein (GFP), isolated from the jellyfish
Aequorea victoria , emits green light when excited by blue or ultraviolet (UV) light, and is by far the most popular fluorochrome used for these applications. Three dimensional (3D) visualisation of gene expression or protein transport in intact specimens has been made possible by the development of confocal and two-photon microscopy
(Gilroy, 1997; König, 2000). These microscopic techniques have a bigger depth of visualisation compared with conventional microscopy. Several reviews are available that provide a detailed overview of recent research in the field of GFP imaging (Haseloff, 1999; Köhler, 1998). GFP imaging does not always allow efficient visualisation.
To avoid problems with high background autofluorescence, double reporter systems could be used, for instance by combining luciferase (see 3.2) and GFP (Mantis and
Tague, 2000).
A recent development with high-throughput potential for in-vivo screening of protein-protein interactions is the protein fragment complementation assay (PCA). The principle of this assay is based on the reconstitution of the 3D structure of native murine dihydrofolate reductase (mDHFR) from two separate but complementary parts of the enzyme, each fused to one of a pair of interacting proteins. Upon interaction of the selected pair of proteins the mDHFR inhibitor methotrexate (MTX), linked to a

fluorescent probe, specifically binds to the reconstituted mDHFR. Protoplasts were transformed by electroporation with plasmid DNA carrying gene fusions encoding the selected interacting proteins, each linked with a fragment of mDHFR. The effect of
(putative) resistance-inducing compounds, added together with MTX to the protoplasts, can be studied at the protein level in vivo, also enabling to elucidate the subcellular location of the interaction (Subramaniam et al., 2001).
In addition to the use of fluorescence imaging (see 5.2) and thermography (see
2.6), confocal microscopy can be used for characterisation of surface and subsurface fruit characteristics in quality assessment. Repeated non-destructive visualisation of the evolution of apple fruit wax layer thickness was recently achieved. The apple cuticular layer prevents water evaporation from the fruit and determines storage time
(Veraverbeke et al., 2001) (see also 5.3).
5.2. Chlorophyll fluorescence imaging
All plant material that contains chlorophyll pigments will emit red fluorescence upon illumination. This chlorophyll fluorescence has an enormous potential as a nondestructive probe to investigate the physiology and structure of the photosynthetic apparatus (Krause and Weiss, 1991). By use of portable fluorimeters, this simple non-invasive means of studying photosynthetic electron transfer reactions has been applied in several fields, as an efficient tool in basic photosynthesis research, to describe and investigate the photosynthetic light processes and quantum conversion at physiological conditions (Govindjee, 1995; Strasser et al., 1995; Stirbet et al., 1998), as well as to detect stress and senescence in the photosynthetic apparatus (Lichtenthaler and Rinderle, 1988; Mohammed et al., 1995; van Kooten and Snel, 1990;
Strasser and Tsimilli-Michael, 1998). However, these instruments have an important limitation. Portable fluorimeters are equipped with small-sized sensors, which deliver measurements of fluorescence intensity averaged over the area of the sensor. The information obtained is limited to a small spot, which is rarely representative for the situation in the whole leaf (Ciscato and Valcke, 1998, Figure 4). Information about the spatial distribution of fluorescence emission can be obtained by performing series of measurements, regularly distributed over the surface to be analysed; this approach however is tedious and inefficient. Over the last decade, high-resolution fluorescence-imaging techniques have been developed. In a sense, the very first experiment about fluorescence imaging can be ascribed to Kautsky and Hirsch (1931) who used their own eyes to visually observe chlorophyll fluorescence changes. The first images of chlorophyll fluorescence were obtained photographically by a technique called phytoluminography (Sundbom and Björn, 1977). The advent of CCD technology and digital imaging techniques allow qualitative and quantitative analysis of fluorescence images. High resolution imaging of chlorophyll fluorescence from intact leaves has enabled the production of images of the relative quantum efficiency of photosynthetic electron transport in tissues, individual cells and even chloroplasts in situ (Oxborough and Baker, 1997; Baker et al., 2001). This technique offers the possibility to study the distribution and irregularities of chlorophyll fluorescence over the whole leaf area (Buschmann and Lichtenthaler, 1998) and enables the analysis of the progressive loss of photosynthetic activity of leaves under stress conditions
(Lichtenthaler and Miehé, 1997). Fluorescence imaging systems have also been used to investigate the role of stomatal conductance in the regulation of photosynthesis (Cardon et al., 1994; Meyer and Genty, 1999), to study the effect of fungal pathogens on photosynthesis (Peterson and Aylor, 1995; Scholes and Rolfe, 1996;
Meyer et al., 2001) and to demonstrate photosynthetic oscillations in leaves (Siebke and Weiss, 1995). The use of chlorophyll imaging in agriculture and horticulture has still been very limited (Abbott, 1999). Only a few attempts have been made to correlate chlorophyll fluorescence emission to physiological properties of fruits in order to estimate maturity and senescence (Song et al., 1997), to predict development of dis-

Figure 4. Chlorophyll fluorescence imaging of the dynamic evolution of abiotic photosynthetic stress induced by the heavy metal copper. A tobacco leaf was darkadapted and subsequently submitted to continuous light. An image of maximum fluorescence (F m
) was captured immediately after this induction. Images of steady state fluorescence (Fs) were captured after induction reached an equilibrium state. The treatment consisted in injecting a copper-sulphate solution in the mid-vein at the base of the leaf, after a control image of the leaf (left panel) was taken. The depicted fluorescence parameter, called ratio of fluorescence decay (Rfd, also variable chlorophyll fluorescence ratio), indicates photosynthetic efficiency and is obtained by the following formula: Rfd=(Fm-Fs)/Fs. Low values (red as indicated on the scale bar) result from a lack of photosynthetic activity. As copper is transported through the vascular system, Rfd values drop, indicating inhibition of photosynthesis (see second panel,
30 min. after treatment and right panel, 60 min after treatment). The left panel shows the distribution of Rfd for a typical untreated leaf region. Figure reproduced with permission from Ciscato and Valcke, 1998. eases (DeEll et al., 1996) or to detect stress conditions during storage (DeEll et al.,
1995). However, these studies used the conventional portable instrumentation and not the fluorescence imaging as such.
Illuminating plant material (e.g. leaves, fruit) with UV-A laser light results in a fluorescence emission spectrum characterised by four emission bands: a blue (440 nm), a green (520 nm) and the well known red (690 nm) and far-red (740 nm) chlorophyll fluorescence emission bands (Chapelle et al., 1984). The blue-green emission, although not yet very well established, relates to secondary metabolites (Goulas et al., 1990) (see also 5.3). High spatial resolution imaging systems have been developed, which allow a fast and large-scale screening of fluorescence gradients and local disturbances in fluorescence emission over the whole leaf surface at the four characteristic emission bands (Chappelle and Williams, 1987; Lang et al., 1994).
Such a system has been used to evaluate nitrogen fertilisation in apple orchards
(Sowinska et al., 1998), to detect physiological disorders during storage of apples
(Ciscato et al., 2001), to study the dynamic evolution of heavy metal stress (Valcke et al., 1999) and as a diagnostic tool for plant stress (Lichtenthaler and Miehé, 1997).
5.3. Accumulation of secondary metabolites
In response to biotic and abiotic stresses, plants accumulate secondary metabolites.
In order to analyse these compounds, new spectroscopic methods, which are nondestructive, rapid and environmentally friendly, compared to the traditional and slower physico-chemical methods have been developed during the last decades. Practically all substances will give rise to substantial absorption and scattering effects when illuminated and can be analysed using vibrational spectroscopies such as near infrared, mid-infrared and Raman. Information on trace substances, present in complex samples such as food, and on their detailed conformation is hidden in their spectra. A limited number of trace substances, which contain one or more fluorophores,

can be detected and measured using fluorescence emission spectra. Most naturally occurring products do not fluoresce, so emission spectra can be measured against a black background. Important fluorophores include the amino acids tryptophan, tyrosine and phenylalanine as protein constituents, coenzymes and nucleotides of the energetic metabolism (NADH, NADPH and FAD), vitamins (A, B1, B2, B6, B12, D2,
E and folic acid), chlorophyll, and secondary metabolites such as caffeine, polyphenols, flavonoids and aflatoxins. Fingerprinting with fluorescence spectroscopy becomes a powerful technique. In this technique, two-dimensional fluorescence landscapes are obtained by measuring the fluorescence spectra as a function of two variables, the excitation wavelength and the emission wavelength (Engelsen, 1997).
Employing ‘Partial Least Square Regression (PLSR)’ techniques, a number of fat- and oil-related quality attributes such as anisidine value, oligomer content, iodine value and vitamine E content can be predicted or measured in complex food samples
(Munck et al, 1998).
In most cases, endogenous fluorophores require excitation with UV light (see also
5.2). Long-term UV irradiation during continuous microscopic observation often leads to photodamage (Cheng et al, 2001) (see also 5.1). To overcome this problem, timeresolved fluorescence microscopy using two-photon excitation offers new opportunities to investigate excited-state dynamics. In fluorescence lifetime imaging (FLIM), the far-field fluorescence decay is recorded with a scanning microscope and an image is constructed from the fluorescence lifetime values obtained for each voxel (volume pixel - picture element) of the scanned field (Draajer et al, 1995). Using FLIM, heterogeneity in the aggregation of LHCII was demonstrated (Barzda et al, 2001). In food analysis, such as quality assessment of fruit, this kind of non-destructive analysis of secondary metabolites seems to be very promising (vande Ven et al., 2001).
5.4. Remote sensing applications
Environmental control of vegetation requires the development of rapid screening techniques, which can be applied on a large scale. Until 1995, remote sensing was almost exclusively carried out by measuring the reflectance signals and images via aircraft and satellites (Nilsson, 1995). On a large scale, fluorescence imaging offers fast and remote measurements from a leaf to a canopy. Nevertheless, since the intensity of actively induced fluorescence is ten fold lower when compared to the reflectance signal, fluorescence images of plants can only be taken from short distances. Moreover, more sophisticated instruments than those for reflectance measurements are needed: a strong source of radiation for exciting the fluorescence and synchronised amplification to discriminate the low-intensity fluorescence from the background of high-intensity daylight reflectance. An overview of the possibilities and limitations in multi-colour fluorescence imaging of plants is given by Buschmann and
Lichtenthaler (1998). Their extension to remote sensing is covered by Cerovic et al.
(1999) (see also 7). The effect of temporal changes in the physical environment of plant communities has been monitored remotely by changes in surface chlorophyll concentration in the oceans and in the Normalized Difference Vegetation Index
(NVDI) on land. The consequences of the El Niño – Southern Oscillations (ENSO) on the biospheric primary production has recently been studied using the Sea-viewing
Wide Field-of-view Sensor (SeaWifs) measuring fluorescence at four different wavelengths (Behrenfeld et al., 2001). Using a newly developed infrared fast repetition rate fluorimeter (IRFRR), the distribution of aerobic bacterial photosynthesis in tropical surface waters and in temperate coastal waters was mapped in a biophysical way
(Kolber et al., 2000). The spatial distribution of photosynthetic bacteria and oxygenic phytoplankton was obtained by measuring the fluorescence emission signals at 880 nm and 685 nm, respectively.

6. MAGNETIC RESONANCE IMAGING
In plant physiology, quantitative analysis of metabolites and modelling of metabolic pathways is still in progress. Firstly, the continuing advances in molecular biology provide tools for dissecting the operation of metabolic pathways and secondly, advances in mathematical modelling and computer programming made analyses of metabolic fluxes more efficient. Another versatile technique to investigate plant metabolism is nuclear magnetic resonance (NMR) spectroscopy (Ratcliffe and Shachar
Hill, 2001). Recording and interpretation of NMR spectra of plant tissue has been described extensively (Roberts and Xia, 1995; Shachar-Hill and Pfeffer, 1996). Twodimensional phosphorus NMR exchange spectroscopy has been used to monitor several reactions of central plant metabolism (Bligny and Douce, 2001). Spatial information from NMR signals can be extracted using NMR imaging (generally referred to as magnetic resonance imaging - MRI). The strongest signal that can be detected in vivo is the 3 H NMR water signal, allowing visualisation of water movement in plants
(Ratcliffe, 1994; Velikanov et al., 2001). Besides describing tissue anatomy and water movement, MRI is also capable of generating physiologically important informa tion (Köckenberger et al., 1997; Köckenberger, 2001). More abundant primary and secondary metabolites have been successfully mapped using imaging methods
(Koizumi et al., 1995; Ishida et al., 1996). The tissue distribution of carbohydrates and amino acids has been mapped simultaneously in hypocotyls of castor bean seedlings using the technique of correlation peak imaging (Ziegler et al., 1996) which allows the detection of the metabolites present at concentrations as low as 10 mM and in a voxel volume of 0.56 µl (Metzler et al., 1995). Although the in vivo histochemistry is still limited by the sensitivity, long-term measurements of both xylem and phloem sap has been performed (Peuke et al., 2001) and chemical shift imaging can generate physiologically important information about the translocation of sucrose, for instance in the phloem (Verscht et al., 1998).
Recently, a positron emitting tracer (PET) imaging system, which has already been widely applied in medical diagnosis, was used to visualise real time translocation of nitrogen ( 13 N labelled) and water ( 15 O labelled) in living rice plants (Kiyomiya et al., 2001). NMR and PET imaging visualise transport phenomena inside plants with sufficient spatial and temporal resolution to study influences of stress factors.
7. MULTISPECTRAL IMAGING
7.1. Multispectral imagers
The term multispectral is generally used to indicate systems that measure reflectance in narrow regions of the visible and near infrared (up to 2000 nm) spectrum (e.g. with
10-nm spectral resolution). The term hyperspectral imaging is also used for such systems. Depending on the wavelength, leaves absorb and reflect more of the captured light. Deterioration of photosynthetic pigments, accumulation of secondary compounds upon a(biotic) stress and structural changes at the leaf surface influence the spectral characteristics of the leaves (Peñuelas and Filella, 1998).
To determine at which wavelength(s) a given (a)biotic stress is most effectively detected, a multispectral imager, which captures data from separate regions of the spectrum (down to 10 nm), is very appropriate. Kobayashi et al. (2001) applied an airborne multispectral scanner to setup a detection methodology for rice panicle blast. Ratios of the signal in different wavelength bands are generally adopted as stress indicators. Airborne digital imagery is one of the key technologies in precision agriculture. Yang et al. (2000) showed that growth data extracted from near infrared images taken during development of a field crop correlated well with the ultimate crop yield. The imaging data thus provides a precision tool to correct for heterogeneity in growth by selective application of fertiliser. The hyperspectral compact airborne spec-

trographic imager (CASI) was used to estimate chlorophyll content in closed forest canopies (Zarco Tejada et al., 2001). At growth stages when soil is still visible as background, the image data corresponding to plant leaves needs to be extracted from the different spectral images (Marchant et al., 2001) (see also 4).
Mutispectral imaging is also applied in machine vision systems for fruit quality control purposes. The imaging system, which combined visual and near infrared radiation (NIR - see also 4) detection with 40nm resolution, was used in conjunction with neural networks for classification of fruit depending on their type and extent of damage (Guyer and Yang, 2000).
7.2. Multiple-detector systems
Combination of multispectral reflectance imaging, chlorophyll and blue/green fluorescence imaging and thermography would provide a platform for the simultaneous noninvasive assessment of multiple physiological parameters. Applications include quantification of the efficiency of developed phytoprotective products or resistanceinducing compounds (for instance acibenzolar-S-methyl). This would complement molecular data, such as those obtained from microarrays (Reymond, 2001) and biochemical information on the influence of these treatments on plant-pathogen systems. A shortening of the necessary monitoring time by presymptomatic quantification of the stress response would be a major contribution to efficient screening programs. Also transgenic plants manipulated for increased stress resistance could be assessed in a more detailed way by simultaneous imaging of multiple characteristics.
Multispectral monitoring systems have the potential for early detection of infected plants. Early assessment of emerging fungal, bacterial or viral infections should prevent further expansion of disease by taking appropriate measures. Identification of the type of stress causing the visualised symptoms would at a first level be derived from the information available from different spectral bands. In the case of biotic stress, a final characterisation by polymerase chain reaction (PCR) of marker genes
(Verdier et al., 2001) may be needed for appropriate treatment. Epidemics of soilborne plant disease could likely be detected in fields, as they appear in because of limited spreading via the soil (Truscott and Gilligan, 2001).
8. FUTURE PERSPECTIVES
Tools for predicting the short-term evolution of plant growth parameters based on weather conditions are receiving increasing interest. Simulation approaches (for a review see Room et al., 1996) are used to model transpiration of canopies using virtual plants (Dauzat et al., 2001). The influence of temperature and other environmental factors on transpiration in the field was shown to be reliably predicted by this simulatory approach.
Robotisation of imaging will enable efficient screening of plant populations
(Chaerle and Van Der Straeten, 2000). This could be applied to screen for mutants in photosynthetic efficiency (Varotto et al., 2000). As an extension to the current efforts in genome characterisation, imaging techniques could also prove to be useful in the elucidation of the function of unknown genes introduced into transgenic plants
(Chaerle and Van Der Straeten, 2001).
Since engineering plants for disease resistance is becoming a reality with the unravelling of the plant genome (Stuiver and Custers, 2001), imaging could be applied to quantify the temporal and spatial characteristics of increased resistance in transformed plants. By applying this method the most promising transgenic plants could be rapidly selected. Another approach made possible by timely detection of (a)biotic stresses by imaging, would consist in switching on engineered stress resistance genes with environment-friendly inducers, which are either volatile or can be applied as a liquid spray (Pickett and Poppy, 2001; Mittler and Rizhsky, 2000).

9. CONCLUSIONS
With the currently available imaging detector technology and portable computing power, (multispectral) monitoring solutions will likely be incorporated into management strategies for plant breeding and ultimately crop production. Such mobile systems could provide an early warning system. Subsequently other measuring techniques need to be used to verify the obtained indications. Fast characterisation of emerging stress has the potential to increase both quality and productivity of agricultural crops.
ACKNOWLEDGEMENTS
We thank M. De Cock for layout. This research was supported by grants from the Fund for Scientific Research (Flanders) (G0068.90, G0021.92, G.0023.95N, 1.5.514.98 and
G0015-01).
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