JXB Advance Access originally published online on November 30, 2006
Journal of Experimental Botany 2007 58(4):797-806; doi:10.1093/jxb/erl208
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Leaf Scale Studies: Combination Imaging and Stress Diagnosis |
Visualization of dynamics of plant–pathogen interaction by novel combination of chlorophyll fluorescence imaging and statistical analysis: differential effects of virulent and avirulent strains of P. syringae and of oxylipins on A. thaliana
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1Julius-von-Sachs-Institute of Biosciences, Department of Pharmaceutical Biology, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
2Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic
3Institute of Physical Biology, University of S. Bohemia, Zámek 136, 37333 Nové Hrady, Czech Republic
To whom correspondence should be addressed. E-mail: benedikty{at}greentech.cz
Received 7 May 2006; Accepted 20 September 2006
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Abstract |
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Pathogen infection leads to defence induction as well as to changes in carbohydrate metabolism of plants. Salicylic acid and oxylipins are involved in the induction of defence, but it is not known if these signalling molecules also mediate changes in carbohydrate metabolism. In this study, the effect of application of salicylic acid and the oxylipins 12-oxo-phytodienoic acid (OPDA) and jasmonic acid on photosynthesis was investigated by kinetic chlorophyll fluorescence imaging and compared with the effects of infection by virulent and avirulent strains of Pseudomonas syringae. Both pathogen strains and OPDA caused a similar change in fluorescence parameters of leaves of Arabidopsis thaliana. The response to OPDA appeared faster compared with that to the pathogens and persisted only for a short time. Infiltration with jasmonic acid or salicylic acid did not lead to a localized and distinct fluorescence response of the plant. To capture the faint early symptoms of the plant response, a novel algorithm was applied identifying the unique fluorescence signature—the set of images that, when combined, yield the highest contrast between control and infected leaf segments. Unlike conventional fluorescence parameters, this non-biased approach indeed detected the infection as early as 6 h after inoculation with bacteria. It was posssible to identify distinct fluorescence signatures characterizing the early and late phases of the infection. Fluorescence signatures of both infection phases were found in leaves infiltrated with OPDA.
Key words: Biotic stress, chlorophyll fluorescence, combinatorial fluorescence imaging, harmonically forced oscillations, jasmonic acid, OPDA, photosynthesis, salicylic acid
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Defence mechanisms are aimed at preventing successful invasion and spreading of micro-organisms attacking the plant or to limit its detrimental effects. Besides non-host resistance against all races of a pathogen, host resistance confers resistance to specific, avirulent strains of a generally virulent pathogen. While the virulent strain is able to spread successfully in the host, the avirulent strain is recognized very early and the plant can effectively defend itself (Baker et al., 1997). One of the defence reactions typical for an incompatible interaction with an avirulent strain is programmed cell death of pathogen-attacked cells—the hypersensitive response (Heath, 2000). In addition, a whole plethora of defence responses such as the accumulation of phytoalexins, cell wall strengthening, and the expression of proteins with antimicrobial properties are induced (Dangl and Jones, 2001). The main difference in the plant responses to a virulent and an avirulent strain is the time-course and the extent of the defence reactions, which are much faster and stronger with the avirulent strain (Tao et al., 2003).
The induction of the defence responses is signalled by salicylic acid, ethylene, and jasmonic acid (Devoto and Turner, 2005). More recently, it was shown that 12-oxo-phytodienoic acid (OPDA), a biosynthetic precursor of jasmonic acid, is also biologically active (Stintzi et al., 2001; Taki et al., 2005). OPDA as well as jasmonic acid are derived from linolenic acid and are ubiquitous signals in the plant kingdom. Endogenous levels of these oxidized lipids increase after pathogen infection (Block et al., 2005). Exogenous application of these compounds induces defence gene expression (Creelman and Mullet, 1997). A strong support for an important role for these signalling molecules in defence against pathogens was provided by the altered susceptibility of mutants defective in oxylipin biosynthesis or signalling (Berger, 2002). The activities of OPDA only partly overlap with those of jasmonic acid, suggesting that OPDA has functions similar to jasmonic acid as well as an independent biological activity of its own. This is supported by the analysis of the mutants dde2, which is not able to synthesize OPDA and jasmonic acid, and opr3, which accumulates OPDA but not jasmonic acid. dde2 but not opr3 is more susceptible to necrotrophic fungi (Raacke et al., 2006). This indicates that OPDA is more important than jasmonic acid in the defence against these pathogens. In contrast, both mutants are more resistant to Pseudomonas syringae, suggesting that in the wild type, both jasmonic acid and OPDA negatively affect the defence against this pathogen. The salicylic acid pathway is the most important contributor to resistance against P. syringae (Delaney et al., 1994). A negative cross-talk between salicylic acid and oxylipins has been reported (Cui et al., 2005). Additionally, other research indicates that, depending on the concentration used, salicylic acid and jasmonic acid can have synergistic effects (Mur et al., 2006).
In addition to defence induction which is generally related to secondary metabolism, pathogen infection induces dramatic changes in the primary metabolism of the affected plant tissue. Several pathogens have been found to suppress photosynthesis of the host (Balachandran et al., 1994; Scholes and Rolfe, 1996; Scharte et al., 2005). In contrast, some pathogens pursue an alternative strategy maintaining photosynthesis of the host in green islands of the leaves (Angrasharma and Mandahar, 1993). With either of these pathogen strategies, the carbohydrate metabolism of the infected plant tissue is substantially modified. The sink strength of the plant is typically increased with assimilates mobilized for defence reactions (Roitsch et al., 2003; Roitsch and Gonzalez, 2004). Also affecting the plant metabolism, biotrophic pathogens drain the host resources by consuming assimilates. A co-ordinated regulation of defence, sink metabolism, and photosynthesis has been observed (Jang and Sheen, 1994; Herbers et al., 1996; Ehness et al., 1997), and sugars and defence-related stimuli were shown to mediate this regulatory mechanism independently (Ehness et al., 1997; Sinha et al., 2002). However, it is not clear whether carbohydrate metabolism and defence responses may also be differentially regulated. In favour of a co-ordinated regulation, jasmonic acid was shown to repress the expression of RbcS (Rubisco small subunit) transcriptionally and post-transcriptionally and to promote fruit ripening, senescence, and tuber formation (Creelman and Mullet, 1997).
The goal of the present study was to contribute to a better understanding of the regulation of photosynthesis in plants attacked by a pathogen. Fluorescence signatures in Arabidopsis thaliana responding to externally administered oxylipins and salicylic acid were being compared with signatures elicited by virulent and avirulent strains of the hemibiotrophic bacterium P. syringae. For this purpose, chlorophyll fluorescence imaging was used with a complex set of conventional (van Kooten and Snel, 1990; Maxwell and Johnson, 2000; Oxborough, 2004) and non-conventional (Nedbal and Brezina, 2002; Nedbal et al., 2003) actinic light protocols. Unique fluorescence signatures were identified by an advanced statistical analysis of the fluorescence image sequences. The technique resolved very early and late phases of the plant response to the pathogen infection as well as revealing differential effects of the signalling molecules.
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Materials and methods |
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Plants
Wild-type A. thaliana Col gl1 was grown with a 9 h light/15 h dark cycle in a controlled environment of a Sanyo Gallenkamp growth chamber (Leicester, UK). The plants were exposed to 150 µmol (photons) m–2 s–1 of white light at 25 °C and at 60% relative humidity during the day. During the night, the temperature was 20 °C and relative humidity 65%. Approximately 6-week-old plants with 10–12 fully developed leaves were used for the experiments.
Bacteria
The virulent bacterial strain P. syringae pv. tomato DC3000 was kindly provided by B Staskawicz, Berkley, CA, USA as well as the avirulent strain that differs by the plasmid carrying the avrRpm1 gene. The bacteria were cultured in Kings B medium containing 50 mg l–1 rifampicin and additionally 10 mg l–1 tetracycline for the avrRpm1 strain. An overnight culture was diluted 1:50 with fresh King's B medium prior to the experiment and cultivated for another 2 h at 28 °C. Bacterial cells were harvested by centrifugation (4 °C, 5000 rpm, 10 min) and dissolved first in 10 mM MgCl2 to optical density (OD)=0.2 measured at 600 nm which corresponded to 
108 cfu ml–1. The final cell concentration of 107 cfu ml–1 was achieved by another dilution. A needleless plastic syringe was used to gently press 10 µl of bacterial suspension into the abaxial side of the leaf. The infiltration was done in the central part of one half of the leaf. The other half of the leaf remained untreated and served as a control. Control plants were inoculated with 10 mM MgCl2. Four leaves of each plant were infiltrated and 2–5 plants were characterized for each of the treatments (virulent and avirulent P. syringae, OPDA, jasmonic acid, and salicylic acid).
Signalling molecules
For treatments with salicylic acid, a solution of the sodium salt obtained from Merck was used. Jasmonic acid was prepared by alkaline hydrolysis of jasmonic acid methyl ester. OPDA was synthesized from linolenic acid using linseed acetone powder and high-performance liquid chromatography (HPLC) purification as described in Parchmann et al. (1997). Oxylipins were diluted to a final concentration of 500 µM in 0.5% methanol before infiltration. Controls were infiltrated with 0.5% methanol, which did not result in effects on photosynthetic parameters at the time points indicated.
Kinetic imaging of chlorophyll fluorescence
Chlorophyll fluorescence imaging was performed with the FluorCam imaging fluorometer (P.S.I., Brno, Czech Republic, www.psi.cz) adapted to generate harmonically modulated actinic irradiance (Nedbal and Brezina, 2002). In order to evaluate contrast between the infected and healthy plant tissue, a complex actinic light protocol was employed that is described in detail in the Supplementary data available at JXB online). The protocol started with 10 min of dark adaptation followed by measuring flashes (620 nm) that yielded images of minimum fluorescence F0 of the plant with a fully oxidized plastoquinone pool (F0). A short saturating flash of 1200–1500 µmol (photons) m–2 s–1 of white light transiently reduced the plastoquinone pool, leading to an increase to the maximum fluorescence (FM). After a short dark relaxation, the first actinic irradiance [50 µmol (photons) m–2 s–1, 620 nm] was applied for 100 s and the corresponding transient of the Kautsky effect was recorded. Two saturating flashes [F'M(1) and F'M(2)] were applied to probe the light-induced non-photochemical fluorescence quenching. The intensity of all saturating flashes applied in the protocol was kept between 1200 and 1500 µmol m–2 s–1 in the field of view. Fluorescence yield was also measured during 15 s of the dark adaptation followed by the actinic light period. Two other saturating flashes [F'M(3) and F'M(4)] were applied to probe the relaxation of the non-photochemical fluorescence quenching. The experiment continued with 600 s of dark adaptation that was followed by an identical sequence of measuring events applied in the actinic light of 200 µmol (photons) m–2 s–1. This actinic irradiance was chosen with respect to the capacity of the measuring instrument to represent the high light level.
The Kautsky transients with the two irradiance levels, I(t)=50 and 200 µmol m–2 s–1, were followed by a battery of transients elicited by harmonically modulated irradiance I(t)=I0+I0·sin(2
·t/T), where T was the period of the harmonic forcing and 2I0 was the amplitude of the actinic light modulation. Details of the technique are described in Nedbal and Brezina (2002), and Nedbal et al. (2003, 2005). Here, fluorescence transients with forcing periods of T=1.5, 3, 6, 12, 24, and 48 s and with two amplitudes 2I0=80 and 160 µmol m–2 s–1 were included.
The a priori feature selection reduced the number of fluorescence images representing the entire fluorescence transient from 727 to 331: 247 in Kautsky effect transients and to 84=2x6x7 images in forced oscillations (Supplementary data available at JXB online). The seven images characterizing the forced oscillation for each period and amplitude of the forcing were the amplitudes and the phases of the fundamental component (period equal to forcing, T) and of two upper harmonics (one-half and one-third of the forcing period, T/2 and T/3), and average fluorescence yield.
These 331 images, each consisting of 512x512 pixels with 12 bit resolution, represented the basic data set characterizing the infection or infiltration response of a single plant. Typically, four attached mature leaves of each plant were treated and 2–5 plants were characterized in each of the treatment phases. Time points analysed were 3, 6, 9, 24, and 48 h after treatment. The selected leaves were gently placed on the black paper masks that hid other leaves and prevented substitution of treated and untreated leaves.
The statistical feature selection and the image segmentation were based on evaluating individual pixels, while individual leaves consisted of 600 pixels.
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Results |
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Effect of the infection of leaves by a virulent strain of P. syringae on conventional chlorophyll fluorescence parameters
A suspension of the virulent strain of P. syringae DC3000 was infiltrated into one of the two halves in each treated A. thaliana leaf. Four mature leaves of each plant were inoculated. Chlorophyll fluorescence transients of the entire plant were imaged before the inoculation, and 3, 6, 9, 24, and 48 h after the inoculation. Figure 1 shows leaf photographs and images of three selected conventional fluorescence parameters, FV/FM, Rfd, and NPQ, in control plants (left column) and in plants 6 h (middle column) and 24 h after the inoculation (right column). The white arrows indicate the infiltrated leaf segments. The false colour scales shown to the right of the fluorescence images indicate the amplitude of the particular parameter.
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No symptoms were detectable by eye 6 h after the P. syringae infection, and only faint symptoms were visible after 24 h as documented by the colour photographs in the top row of Fig. 1. The FV/FM fluorescence parameter shown in the second row of Fig. 1 is interpreted as the maximum quantum yield of photosystem II (PSII) (Buffoni et al., 1998; Genty et al., 1989). It remained uniformly high, close to 0.8, 6 h after the inoculation (middle image). A localized decline of FV/FM was documented 24 h after the inoculation with bacteria. This was in agreement with an earlier report on a localized, infection-induced decrease in photosynthesis (Bonfig et al., 2006). The third row in Fig. 1 documents pathogen-induced dynamics in another parameter that is interpreted as a measure of photosynthetic capacity, i.e. the fluorescence decrease ratio Rfd=(FP–FS)/FS (Lichtenthaler and Miehe, 1997; Lichtenthaler and Babani, 2000; Lichtenthaler et al., 2005a, b). In control plants, the Rfd parameter was significantly less uniform than FV/FM (first images from the left). The Rfd heterogeneity was further increased 6 h after the inoculation (middle image). The trend was not clear though, with a decrease of Rfd in the area of infection in half of the imaged leaves and with a faint increase in the remaining half (Fig. 1). The Rfd parameter was clearly depressed in the infected leaf segment 24 h after the inoculation with bacteria. The bottom row of the fluorescence images represents the dynamics of non-photochemical quenching, NPQ=(FM–F'M)/F'M (Horton and Ruban, 1992). An increase of NPQ in the area of infection was seen to various extents already after 6 h (middle image). Although the NPQ seemed to be more responsive to the infection than both FV/FM and Rfd at this time point, the reliability of this parameter was, however, reduced by its intrinsic leaf-to-leaf variability and by its sensitivity to the geometrical arrangement of the leaf with respect to the incident saturating light (patches in the control plant in Fig. 1). The other source of NPQ variability could be the intensity of the saturating flash that decreased towards the margins of the field of view from 1500 to 1200 µmol (photons) m–2 s–1. The heterogeneity of the NPQ response was further increased 24 h after the inoculation (right image). In some leaves, the NPQ was decreased in the core of the infected leaf area, whereas it was increased around it. This variability of NPQ lowers the potential of this parameter to serve as a reliable classifier of the infection.
Differential effects of oxylipin treatment and P. syringae infection on chlorophyll fluorescence signatures
Figure 1 shows that the infection differentially modified the three selected fluorescence parameters. Figure 2 shows the variability of the fluorescence response for a more extended set of conventional fluorescence parameters (Rohacek, 2002). The three spider plots show the dynamics of 19 conventional fluorescence parameters during the infection of A. thaliana by the virulent DC3000 (Fig. 2A) (Table S1 in the Supplementary data available at JXB online) and avirulent RPM1 (Fig. 2B) (Table S2 in the Supplementary data available at JXB online) strains of P. syringae and by infiltration with OPDA (Fig. 2C) (Table S3 in the Supplementary data available at JXB online). Each spider plot consists of three segments: the dark-shaded segment represents the parameters measured with dark-adapted plants, the light-shaded segment corresponds to the Kautsky induction measured in low light (50 µmol m–2 s–1), and the segment without shading represents parameters measured in high actinic light (200 µmol m–2 s–1). The distance from the centre of the spider plot indicates the relative change of the fluorescence parameter during the treatment. The change was measured by comparing the signals in the untreated half of the leaf with the leaf segment close to the application site. The values between the centre (–100%) and the solid black circle (0%) represent a decrease of the particular parameter during the infection. The values further to the perimeter (+200%) indicate an increase of the fluorescence parameter. Clearly, the OPDA infiltration elicited in 3 h a response (red line in Fig. 2C) that was similar to the fluorescence response observed in the plants infected by both strains of P. syringae 24 h after the infiltration (red line in Fig. 2A, B). The similarity of fluorescence changes of the three represented treatments indicates with a high confidence that their impact on photosynthetic processes is of a similar biological nature.
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However, the time-course of the fluorescence changes was different with pathogens and with OPDA. The differences in the dynamics of the response to virulent and avirulent strains were small (compare the left and middle spider plots in Fig. 2). In line with expectations, the changes (green line) occurring 6 h after the infiltration were larger in certain parameters with the avirulent strain than with the virulent one. Yet, this faster onset of the response to the avirulent strain was not obvious any longer in plants 24 h after the inoculation (red line). In contrast to the infected plants, the symptoms rapidly disappeared in the OPDA-treated plants and were not observed 6 h after the infiltration (line not shown because of coincidence with the black line in Fig. 2C). The transient character of the OPDA-induced response can be caused by diffusion and dilution of the signalling compound in the plant tissue.
The changes of individual fluorescence parameters represented in the spider plots of Fig. 2 can be characterized approximately into three classes,
- (i) The parameters measured in dark-adapted plants (F0/FM, FV/FM, and FV/F0). The conventional parameter FV/FM interpreted as the maximum quantum yield of PSII exhibited a small decline with all treatments. The other mutually dependent parameters F0/FM and FV/F0 lack a clear physiological interpretation but were, in various forms, shown to yield a high contrast in revealing biotic and abiotic stresses (Nedbal et al., 2000; Soukupova et al., 2003). Also here, F0/FM dramatically increased and FV/F0 dramatically decreased during all the three treatments that are represented in Fig. 2.
- (ii) The quantum yields of non-photochemical processes in PSII in light-adapted plants (
N = F'0/F'M) increased very significantly during the treatment regardless if measured during the first [N(1)] or second of the saturating flashes [N(2)]. The relative change was always higher in the low light (shaded background) than in the high light (no shading). The other fluorescence parameter, the NPQ [NPQ=(FM–F'M)/F'M] is shown in Fig. 1 and discussed in the previous section.
- (iii) The other parameters shown in Fig. 2 stagnated or decreased upon the treatment. The parameters
P(2), 'PSII(2) decreased more during induction in high light (unshaded sectors of the spider plots), whereas the fluorescence decrease ratio Rfd exhibited a significant decline in low light (shaded sectors of the spider graphs).
- (ii) The quantum yields of non-photochemical processes in PSII in light-adapted plants (
The spider plots in Fig. 2 can be considered as fluorescence fingerprints of the stress-induced changes in the photosynthetic processes induced by the individual treatments. The similarity of all three fluorescence fingerprints points to identical molecular mechanisms occurring with different dynamics upon infection of A. thaliana by virulent and avirulent strains of P. syringae and upon infiltration of leaf tissue with OPDA.
The effects of infiltration by jasmonic acid and salicylic acid were also investigated, but no change in the measured parameters exceeded noise. This observation indicates that there were no effects on fluorescence emission, or that the effects were too transient to be captured, or that the effects were systemic rather than localized.
Combinatorial fluorescence imaging
The selection of 19 parameters showing a fluorescence fingerprint (Fig. 2) represents those described earlier in the fluorescence literature (reviewed in Rohacek, 2002). These are typically used as standard parameters for the evaluation of chlorophyll fluorescence data. However, these are not necessarily the parameters that would yield the highest contrast between treated and untreated leaf tissue.
The problem of optimal selection of contrast-yielding fluorescence images is addressed in Matous et al. (2006). Here, a complex experimental protocol was applied with 331 independent fluorescence images capturing Kautsky induction and quenching analysis in 50 and 200 µmol m–2 s–1 and six transients in fluctuating, harmonically forced light (T=1.5, 3, 6, 12, 24, and 48 s) with two amplitudes 80 and 160 µmol m–2 s–1 (Supplementary data available at JXB online). By considering the complete set of fluorescence images and by Sequentional Forward Floating Search (Pudil et al., 1994), one can identify eight fluorescence images that yield the highest contrast between the infected and healthy leaf tissue (Supplementary data available at JXB online). The search can be trained using leaf tissue in the late phase of the infection (by the 24 h trained Late classifier) and, separately, using leaves in the early infection phase (6 h and 9 h trained Early classifier). Figure 3 shows how the algorithm classified individual pixels in fluorescence images of A. thaliana infected by the virulent strain (top block of Fig. 3, left) and the avirulent strain of P. syringae (top block of Fig. 3, right). The bottom figure block shows the classification for infiltration with OPDA (Fig. 3, bottom left) and with jasmonic acid (Fig. 3, bottom right). White shading indicates that the pixel was classified with confidence as belonging to the tissue in the early (left column) or late (right column) phase of the infection by the virulent strain. Black shading indicates that the pixel was classified as healthy tissue.
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Figure 3 shows convincingly that the resolving power of the combinatorial fluorescence imaging significantly exceeds that of conventional fluorescence parameters in Figs 1 and 2. The classifier trained with leaves that were infected by the virulent strain of P. syringae for 6 h and 9 h was able to identify the infected tissue as early as 6 h after the infection by both virulent and avirulent strains. The symptoms were stronger in the avirulent strain than in the virulent strain. With advancing infection, the Early classifier faded away from the centre to the edge of the infected segment, indicating the early phase of the infection at the forefront. The Late classifier trained with leaves that were infected by the virulent strain of P. syringae for 24 h failed to recognize the early infection phase, while it was very powerful 24 h and 48 h after inoculation.
The fact that the classifiers trained with the virulent strain succeeded in recognizing correctly the infection by the avirulent strain confirms the earlier conclusion. The fluorescence signatures and thus the underlying effects on biochemical and/or molecular-genetic processes involved in photosynthesis are identical for both strains (Figs 2, 3).
Figure 3 also shows that the classifiers trained with the virulent strain of P. syringae can detect the effects of OPDA (left bottom) too. This proves that the signature is similar. The fluorescence signature of the infection was found sharply 3 h after the OPDA infiltration by the Late classifier and was only slightly weaker when identified by the Early classifier. The signature disappeared rapidly with only faint residues 24 h after the infiltration.
The combinatorial imaging failed to detect any symptoms when the leaf was infiltrated by jasmonic acid (bottom right) and salicylic acid (not shown). This finding excludes one of the alternative explanations suggested above—a systemic response to the applied plant hormone would be detected as an ‘all white’ image in Fig. 3.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Chlorophyll fluorescence imaging allows monitoring of the spatial distribution of photosynthetic activity and capacity. In addition, this non-invasive method provides the opportunity to measure temporal changes and kinetics of processes affecting photosynthetic performance. In this study, the method was applied to detect biotic stress responses and to analyse changes in photosynthesis in the interaction of the model organism A. thaliana with a virulent and an avirulent strain of P. syringae. Additionally, the changes upon pathogen infection were compared with the effect of the signalling molecules jasmonic acid, OPDA, and salicylic acid.
Kinetic chlorophyll fluorescence imaging is a well-established tool to image localized plant stress (reviewed in Nedbal and Whitmarsh, 2004). The investigated plant is dark-adapted, and the Kautsky-type fluorescence transient is elicited by sudden exposure to constant light and measured together with quenching analysis by saturating light flashes (Schreiber et al., 1986). Frequently, fluorescence parameters used conventionally in plant physiology are scanned and the one yielding the best contrast is used to detect the localized stress (Balachandran et al., 1997; Nedbal et al., 2000; Chaerle and Van der Straeten, 2001; Meyer et al., 2001; Soukupova et al., 2003; Berger et al., 2004; Chaerle et al., 2004). Here, a different strategy was used. A complex protocol was applied including Kautsky-type induction with two different irradiance levels and measurement of fluorescence dynamics in fluctuating, harmonically modulated light of various periods and amplitudes. The protocol is described in the Supplementary data available at JXB online. From this measurement, the conventional fluorescence parameters (Figs 1, 2) as well as 331 independent images were obtained. These images were submitted to a Sequentional Forward Floating Search (Pudil et al., 1994) for eight images that in combination yielded the highest contrast between the healthy tissue and tissue infected by the virulent strain of P. syringae. This combinatorial fluorescence imaging was shown to detect the infection as early as 6 h after the inoculation (Fig. 3), and the results indicate an even earlier detection (unpublished data). The technique also discriminated between the early and the late phases of the infection (Fig. 3).
The non-biased type of analysis to investigate and predict effects on photosynthesis by combinatorial fluorescence imaging (Fig. 3) was shown to be superior to the conventional approaches (Figs 1, 2) in identifying leaf segments that are affected by the infection, as well as in its capacity to discriminate between the early and late phase of the infection. In contrast, imaging of the conventional fluorescence parameters has the great advantage of physiological interpretation. The FV/FM images shown in Fig. 1 reveal the effects of the infection only very late, after 24 h, but their interpretation provides a valuable insight: the capacity of PSII was locally reduced by the infection.
Photosynthesis decreased after treatment with both P. syringae strains. A decrease in photosynthesis, measured mostly as effective PSII quantum yield, has also been reported upon infection by biotrophic as well as necrotrophic pathogens in different plant systems (Balachandran et al., 1997; Nedbal et al., 2000; Chaerle and Van der Straeten, 2001; Meyer et al., 2001; Soukupova et al., 2003; Berger et al., 2004; Chaerle et al., 2004). This comprises compatible as well as incompatible interactions. In contrast, NPQ has been shown to be positively and negatively affected by pathogen attack. The effect on NPQ might depend on the amount of tissue damage. In tissue not heavily damaged, NPQ might increase based on stimulated electron flow as a protection mechanism (Schreiber, 2004). On the other hand, in severely damaged tissue, inhibition of photosynthetic electron transport might result in decreased NPQ.
Treatment with OPDA, but interestingly not with jasmonic acid or salicylic acid, resulted in a decrease in photosynthesis. OPDA is a reactive molecule, since it contains an 
,ß-unsaturated carbonyl structure (Almeras et al., 2003) while jasmonic acid and salicylic acid do not. This difference could explain why OPDA but not jasmonic acid or salicylic acid affected photosynthesis. It has been shown previously that different sugars, which can function as signalling molecules, decrease effective PSII quantum yield (Sinha and Roitsch, 2001). The present study demonstrates that the plant-derived lipid signalling molecule OPDA also exerts similar effects on chlorophyll fluorescence. Comparison of the fluorescence signature after treatment with both P. syringae strains and with OPDA revealed similarities of OPDA treatment 3 h after infiltration with P. syringae treatment 24 h and 48 h after infiltration. This observation is in agreement with an accumulation of OPDA 24 h after P. syringae infection (Block et al., 2005) and leads to the conclusion that OPDA might be involved in the down-regulation of photosynthesis caused by P. syringae. The fast decrease in FV/FM in response to OPDA infiltration indicates a direct effect of OPDA on PSII due to its reactivity (see above). The effects of OPDA treatment were transient, which might be due to diffusion and/or metabolism of the oxylipin. It is possible that within a few hours OPDA is converted to jasmonic acid, which is not active in changing photosynthetic parameters. Changes in photosynthesis were detectable earlier with the avirulent strain which induced stronger responses at the 6 h time point compared with the virulent strain. This is in agreement with the earlier detection of and defence induction against the avirulent strain (Dong et al., 1991; Baker et al., 1997).
The non-invasive analysis of plant primary metabolism by chlorophyll fluorescence imaging has particularly enriched stress physiological studies. The combination with other non-invasive techniques such as imaging of spontaneous photon emission (Bennett et al., 2005), and the detection of fluorescence reporter genes, labelled pathogens, and secondary metabolites should further upgrade the informative value of the approach. The present study demonstrates that both improvement of measurement protocols and optimization of the image analysis are important targets to be able to define fluorescence signatures that eventually allow discrimination between abiotic stress types and different pathogens. This will also be a prerequisite for online measurement in agricultural application for automation of fertilization and pest control.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Numerical values of fluorescence parameters as well as a description of combinatorial imaging, complex actinic light protocols, and Euclidian distance are provided as Supplementary data associated with this article and are available at JXB online.
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Acknowledgements |
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This work was supported in part by the Czech Ministry of Education, Sports, and Youth under the Grant MSM6007665808, by the Czech Academy of Sciences Grant AV0Z60870520, by the Grant Agency of the Czech Republic GACR 206/05/0894, and by the co-operative grants DAAD/CAS D28-CZ31/04-05 (TR) and the SFB 567 (SB). We are grateful to Sigrid Lux for her excellent technical assistance.
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Footnotes |
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* These authors contributed equally to this work.
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Abbreviations |
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F, chlorophyll fluorescence yield (subscripts 0, M, V, P, S define minimal, maximal, variable, peak, and steady-state level, whereas a prime indicates that the parameter was measured in light-exposed plant); FV/FM, maximum quantum yield of PSII photochemistry;
P=FV'/FM', the efficiency of excitation energy capture by open PSII in the light-adapted state; N=1–P=F0'/FM', quantum yield of non-photochemical processes in PSII in the light-adapted state; PSII=(FM–FS)/FM, quantum yield of PSII (non-cyclic) electron transport; 'PSII = (FM'–FS')/FM', effective quantum yield of PSII; NPQ=FM/FM'–1, non-photochemical chlorophyll fluorescence quenching; PSII, photosystem II; Rfd=FP/FS–1, chlorophyll fluorescence decrease ratio; qA=1–F0'/FM, absolute quenching of ChlF; OPDA, 12-oxo-phytodienoic acid. |
References |
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