Correlative Analysis of Fluorescent Phytoalexins by MassSpectrometry Imaging and Fluorescence Microscopy in GrapevineLeavesLoïc Becker,*,† Sebastien Bellow,‡ Vincent Carre,*,† Gwendal Latouche,‡ Anne Poutaraud,§,∥

Didier Merdinoglu,⊥,# Spencer C. Brown,○ Zoran G. Cerovic,‡ and Patrick Chaimbault†

†Universite de Lorraine. Laboratoire de Chimie et Physique-Approche Multi echelle des Milieux Complexes (LCP-A2MC), EA 4632,Institut Jean Barriol − Federation de Recherche 2843; ICPM 1, Boulevard Arago, Metz Technopole Cedex 03, F-57078, France‡Ecologie Systematique Evolution, Univ. Paris-Sud, CNRS, AgroParisTech, Universite Paris-Saclay, 91400, Orsay, France§INRA, Laboratoire Agronomie et Environnement, UMR 1121, Colmar, 29 rue de Herrlisheim, F68021 Colmar Cedex, France∥Universite de Lorraine, Laboratoire Agronomie et Environnement, UMR 1121, 2 Avenue de la foret de Haye - TSA, 40602 - F54518Vandœuvre-les-Nancy Cedex, France⊥INRA, UMR 1131, SVQV, F-68000 Colmar, France#Universite de Strasbourg, UMR 1131, SVQV, F-68000 Colmar, France○Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Universite Paris-Saclay, 91198, Gif-sur-Yvettecedex, France

ABSTRACT: Plant response to their environment stresses isa complex mechanism involving secondary metabolites.Stilbene phytoalexins, namely resveratrol, pterostilbene, piceidsand viniferins play a key role in grapevine (Vitis vinifera) leafdefense. Despite their well-established qualities, conventionalanalyses such as HPLC-DAD or LC-MS lose valuableinformation on metabolite localization during the extractionprocess. To overcome this issue, a correlative analysiscombining mass spectroscopy imaging (MSI) and fluorescenceimaging was developed to localize in situ stilbenes on the samestressed grapevine leaves. High-resolution images of thestilbene fluorescence provided by macroscopy were supple-mented by specific distributions and structural informationconcerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent andcomplementary data on the stilbene spatial distribution for the two stresses addressed: UV−C irradiation and infection byPlasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesisof stilbenes is induced after UV−C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cellwalls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins ofvarious plant tissues with resolution approaching the cellular level.

Studies of secondary metabolites are a key to understandhow plants respond to their environment, to stress and

what mechanisms are involved. Stilbenes are phytoalexinsproduced in the phenylpropanoid pathway and are synthesizedunder biotic stress.1−3 The downy mildew disease, caused bythe oomycete Plasmopara viticola, is one of these whose effectsare well described.4 Identified as phytoalexins of grapevine forthe first time by Langcake and Pryce,5 stilbenes are known fortheir antifungal activity.6−10 Still, their effect on P. viticolamycelia remains a matter of debate.6 Therefore, study ofstilbenes in vivo would contribute to the understanding of thehost−pathogen relationship. After P. viticola infection, grape-vine leaves synthesize trans-resveratrol (3,5,4′-trihydroxystil-bene), trans-pterostilbene (3,5-dimethoxy-4′-hydroxystilbene),

trans- and cis-piceid (3-O-β-D-glucoside of resveratrol), andcyclic dehydrodimers of resveratrol trans-ε-viniferin and trans-δ-viniferin.11−16 Moreover, stilbenes can also be synthesized afterabiotic stress such as UV−C irradiation,17 wound, dryness orchemicals.18 For their investigation, analytical techniques suchas gas or liquid chromatography with UV and massspectrometry detectors are traditionally used on a plant tissueextract.19,20 With these approaches, high levels of resolutionand sensitivity are reached,21 but compound locations in tissuesare lost. Indeed, prior to the analysis, these techniques require

Received: March 17, 2017Accepted: June 1, 2017Published: June 1, 2017

Article

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© 2017 American Chemical Society 7099 DOI: 10.1021/acs.analchem.7b01002Anal. Chem. 2017, 89, 7099−7106

solvent extraction of the sample. This procedure homogenizesthe molecular content of the sample. The mechanismsregulating stilbene synthesis appear complex;6 moreover, theirlocation during the interaction with a pathogen or anotherstress is critical.Imaging techniques may provide metabolite distribution on

the sample surface. Fluorescence imaging of stilbenes ingrapevine leaves is based on their autofluorescence under UVlight, as for several other phenolic compounds.22 Their violet-blue fluorescence (VBF) emission is centered around 390 nmboth in methanol and in leaves. The maximum of excitation isaround 320 nm.23 Although a difference in fluorescence yieldexists among the stilbenes produced by grapevine leaves, theirfluorescence spectra are too similar to be used fordiscrimination in vivo.23,24 Stilbene localization has beenstudied by confocal fluorescence microscopy on leaves ofgrapevine genotypes with different levels of resistance to P.viticola.24 This technique enables in vivo visualization ofphenolic compounds inside leaves by 3D reconstructions andoptical sections.25 Moreover, fluorescence microscopy isnondestructive. Indeed, fluorescence imaging allows theobservation of stilbenes in vivo,23,24 enabling kinetic studieson attached leaves. High resolution images of grapevine leaveshave been obtained but without distinction between thedifferent stilbenes.23,24,26

Through mass spectrometry imaging (MSI), in situcompound identifications can be obtained at the same timeas their surface area distributions. MSI dealing with plantmetabolites has clearly emerged.27−31 Several metabolitefamilies have been observed, such as agrochemicals in soya,32

carbohydrates in wheat stems33 or wheat seeds,34 amino acidsand phosphorylated molecules in wheat seeds,35 or even lipidspresent in an Asian variety of rice resistant to drought.36 Goto-Inoue et al. observed the location of the γ-aminobutyric acid ineggplants.37 Other recent MSI studies of plant tissue deal withtoxic glycoalkaloids in potato tuber,38 symbiosis of plants withnitrogen fixing microorganisms,39 anthocyanins in ricepericarp,40 and glucosinolates in Arabidopsis flowers andsiliques.41 Parallel analyses in mass spectrometry wereperformed on grape berries by Berisha et al. using laserdesorption followed by electrospray ionization (LD-ESI),MALDI imaging, and HPLC/ESI-MS.42 This combinedapproach led to the localization of specific metabolites on theberry surface in addition to the characterization of severalanthocyanins, amino acids, and carbohydrates. Our previousreports of stilbene imaging were performed on grapevine leaveswith laser desorption/ionization (LDI)43 and matrix assistedlaser desorption/ionization (MALDI).44

The present study assesses the feasibility to map plantmetabolites combining MSI and fluorescence imaging. Laserdesorption/ionization mass spectrometry imaging (LDI-MSI)allows characterizing and localizing stilbenes. However, MSIsuffers from low spatial resolution, depending upon the laserfeatures. The best spatial resolutions attained have ranged from5 to 20 μm.45 Moreover, the LDI is a tough destructive process,which may result in high specificity of the ionized compounds.On the other hand, fluorescence imaging produces high-resolution images without damaging the sample. However,these two imaging techniques do not provide quantitative datain absolute values. The good correlation between VBF ofstilbenes and total stilbene content shown by Poutaraud et al.23

is only valid at the macroscopic scale. In microscopic images,the intensity of stilbenes’ VBF cannot be used as a direct

correlation to quantify stilbenes because of the major influenceof the rigidity of stilbene molecules’ environment over theirfluorescence yield and the large differences in the rigidity of thevarious tissue compartments.24 MSI provides molecular mapsfor each ion detected with semiquantitative data in relativevalues. Indeed, the intensity of the pixel is proportional to thenumber of molecules of stilbenes from which the ion is issued.The relative value scales are thus specific to each ion and toeach sample. To overcome this, HPLC coupled to a diode arraydetector (DAD) was used to add quantitative data in absolutevalues and to validate the compound identification of stilbenes.To test this approach, stressed grapevine leaves were studied. Afirst experiment was carried out on grapevine leaves treated byUV−C, which provokes stilbene synthesis on the whole treatedsurface.43 A second experiment was then conducted on leavesinfected by downy mildew.

■ EXPERIMENTAL SECTION

Reagents. Standard compounds of trans-resveratrol, trans-pterostilbene, trans-piceid, δ- and ε-viniferin, and poly(-ethylene glycol) (PEG 600) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Trans-piceid, δ-viniferin, and ε-viniferin were prepared in methanol at aconcentration of 10−4 M. For the LDI-TOFMS analysis ofstandards, 2 μL of each stilbene solution was deposited on thetarget.

Plant Material. Hybrid genotypes of grapevines susceptibleto P. viticola were studied. These hybrids resulted fromcrossings of the American species Muscadinia rotundifolia withVitis vinifera cultivars. Plants were grown from green cuttings inColmar (France) at 22 ± 3 °C with 13/11 light/dark in thegreenhouse. The sixth leaf, counted from the apex of 3.5-month-old plants having 12−14 fully expanded leaves, wassampled and washed with demineralized water.

Leaf UV−C Irradiation. The abaxial side of leaves wasexposed to UV−C radiations at 254 nm (UV−C tube, Osram,30 W, 90 μW cm−2) at 13 cm distance from the lamp. Thefollowing protocol was applied to generate three different zoneson the same sample: control (not irradiated), irradiated for 45s, and irradiated for 180 s. Two covers (i and iii) separated by 2mm (ii) were positioned on the leaf (Figure 1a). After 135 s ofUV−C irradiation, the left cover (i) was removed. Then the leafwas further irradiated for 45 s. The middle area between thetwo covers (ii) was thus irradiated for 180 s. To allow thebiochemical response to develop, the treated leaf was then

Figure 1. Protocols for (a) UV−C irradiation: (i) 45 s irradiated, (ii)180 s irradiated, and (iii) control area (nonirradiated); and (b) P.viticola infection of grapevine leaves. Three dots were deposited oneach leaf with a felt-tip marker. They are used as reference marks toindicate the area to analyze by fluorescence microscopy and by massspectrometry imaging.

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maintained for 3 days in a closed Petri dish with its adaxial sidepressed against wet paper before fluorescence imaging.Leaf Infection by P. viticola. P. viticola was obtained from

naturally infected plants in Colmar (France). Sporangia wereperiodically grown in order to prepare inoculants. The leaf wasinfected by spraying an inoculum solution at a concentration of3 × 105 sporangia/mL on the upper half of the abaxial side ofthe leaf. The lower half of the leaf was protected with a cover, asdescribed in Figure 1b. During and after spraying, the leaveswere put in 14 cm diameter Petri dishes with the adaxial sidepressed against wet paper. The Petri dishes were closed justafter spraying to maintain the leaves under moist conditions tofavor inoculation and sporulation. Analyses were done after 4days of incubation (4 dpi).Fluorescence Imaging (Macroscopy). Images were

acquired using a macroscope (AZ100 Multizoom, Nikon,Champigny-sur-Marne, France) equipped with a 130 W metalhalide lamp white source (Intensilight, Nikon) and a high-resolution color camera (Ds-Ri, Nikon) at room temperature(19 °C). Macroscopy, as opposed to microscopy, ischaracterized by large object fields and large working distances,plus panning and zooming, allowing fluorescence imaging atorgan, tissue, and multicellular levels. The UV-suppression filterof this source was removed. The images of UV-excited visibleautofluorescence were recorded using a custom-made filterblock from AHF (Tubingen, Germany) with an excitationbandpass filter 340/26 (FF01 Brightline, Semrock, Rochester,NY, U.S.A.), a dichroic filter Q380LP (Chroma TechnologyCorp., Bellows Falls, VT, U.S.A.), and a long-pass 371 nmemission filter (LP02−364RS, Semrock). The images of blue-excited green autofluorescence were recorded using a GFP-Bfilter set (excitation band-pass filter 472/30, dichroic filter 495nm, and emission bandpass filter 520/35, Nikon). A ×2objective (NA 0.2, working distanced 45 mm, AZ-Plan Fluor,Nikon) was used and 24-bit RGB color images were acquiredwith a 1284 × 1024 pixel resolution. Imaged leaf pieces wereflattened (abaxial side facing the objective) on the glass sampleholder (adaxial side lightly moistened for adhesion). Theflatness of the imaged area was necessary for a good-qualityacquisition. When present, sporangiophores were washed fromthe sporulating leaves to avoid their contribution to VBF. Imageacquisition was performed using the NIS-Elements software(Nikon). Image analysis, including composition, was performedusing the software ImageJ (http://rsbweb.nih.gov/ij/). For theimages of specific blue-excited green autofluorescence, only thegreen channel of the RGB pictures acquired was used,visualized with a black and green intensity scale (namely,look-up table - LUT). For the images of overall RGB UV-excited visible autofluorescence, images were processed byoptimizing the brightness and contrast in each of the threecolor channels before making RGB overlays. This was necessaryfor a good and simultaneous visualization of both thechlorophyll fluorescence (red channel) and the bluefluorescence (blue channel and slightly green channel) thatincludes stilbene VBF.Confocal Fluorescence Microscopy and 3D Image

Reconstruction. The confocal microscope (LSM510 Meta,Zeiss, Jena, Germany) had an argon laser providing a 488 nmbeam dynamically filtered by an acousto-optic tunable filter(AOTF) that was used to excite the grapevine-leaf greenautofluorescence. All experiments were performed with a ×63objective (Plan-Apochromat, NA 1.40 oil, Zeiss) at roomtemperature (19 °C). The dichroic filter used was HFT UV/

488 (Zeiss). Leaf samples were mounted in oil for microscopy(Immersol 518N, Zeiss) with the abaxial side facing theobjective. The coverslips thickness was 0.170 mm (#1.5).The array detector of the Zeiss LSM510 Meta is a

spectrograph dispersing emitted fluorescence from 361.8 to704.2 nm on a 32 photomultiplier tube (PMT) array. The 32signals were selectively binned for standard imaging. Theimages presented in this paper are the overlay of two detectionchannels on the Meta array detector: 500.9−597.2 nm (greenchannel) and 629.3−682.8 nm (red channel). Series of XYimages, called Z-stack, were acquired along the Z axis, the axisperpendicular to leaf surface and parallel to the excitation beam.The optimal voxel size of 0.26 × 0.26 × 0.63 μm for x, y, and zdirections, respectively, was used. The resolution of theacquired images was 512 × 512 pixels, coded in 8 bits foreach color channel. The Z-stacks allowed a 3D analysis that isshown here through 3D projections. Image acquisition wasperformed using the software Zen (Zeiss). Image analysis,including 3D reconstruction, was carried out using the softwareLSM Image Browser (Zeiss) and the software ImageJ (http://rsbweb.nih.gov/ij/).

Mass Spectrometry Imaging. A Bruker Reflex IVMALDI-TOF mass spectrometer (Bruker Daltonics, Bremen,Germany) was used to perform imaging experiments and toanalyze standards, at room temperature (19 °C). In addition tothe original nitrogen laser (337 nm, Science Inc., Boston, MA,U.S.A.), a second optical pathway into the ionization chamberwas developed in our laboratory that enabled us to performLDI-MS experiments at 266 nm by coupling a quadrupledNd:YAG laser (Continuum, Santa Clara, CA, U.S.A.). Positivemass spectra were acquired in the m/z 0−1000 range. The massspectrometer was operated in the reflectron mode at a totalacceleration voltage of 20 kV and a reflecting voltage of 23 kV.A delay time of 200 ns was used prior to ion extraction. Thelaser energy was kept at 60% of its maximum value (fluence ≈0.5 J/cm2). The laser had a pulse duration of 5 ns and was usedat a repetition rate of 9 Hz. Mass spectra obtained for each pixelcorresponded to the averaged mass spectrum of 50 consecutivelaser shots on the same location. The laser spot diameter wasmeasured at 45 μm; therefore, spatial resolution was fixed at 50μm. FlexImaging software (v.2.1, Bruker Daltonics, Bremen,Germany) was used to perform mass spectrometry imagingexperiments. PEG 600 (10−2 M) was used to perform externalcalibration. Approximately 12 h were required to achieve animage of about 7000 pixels.

Methanolic Extraction and HPLC-DAD Analysis. Theextraction protocol was derived from the method used by Pezetet al.14 Foliar discs of 2 cm diameter were collected close to theimaged area for each condition: control (neither irradiated norinoculated), irradiated for 45 s, irradiated for 180 s, and infectedareas. These were placed in 1 mL of methanol for extraction. Aratio of dry matter to solvent volume less than 15 mg/mL wasmaintained for each extraction. Samples were then placed in awater bath at 60 °C for 45 min with stirring. The extracts werecentrifuged and stored at −20 °C before HPLC analysis.Stilbene quantification was performed with a 1100 HPLCsystem (Hewlett-Packard, Agilent Technologies, Massy,France) equipped with a diode array detector (Hewlett-Packard, 190−950 nm). Separation was carried out on a RP-18 “end-capped” 5 μm 130 Å column (LiChrospher, Merck,Lyon, France) of 250 mm length and 4.6 mm inner diameter,thermostated at 20 °C. The solvent system was the onedescribed by Pezet et al.14 Chromatograms were recorded at

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307 nm. Contents are expressed as means of six replicates ±standard deviations. Statistical analysis was conducted using theR software (v3.2.5, R Core Team). Mean values were comparedby using Student’s t test at p < 0.05 significance level.Handling for Correlative Analyses. Samples were imaged

first by fluorescence and second by LDI-MSI. Immediately afterfluorescence imaging, samples were freeze-dried between twomicroscope glass slides covered by tape to avoid damage(warping and cracking). This fixed the sample state, so wecould observe the same sample with both imaging techniques.Three corners were marked with a felt-tip pen to define thezone selected for imaging (Figure 1). Molecular maps wereprocessed and extracted with the FlexImaging software. Todetermine the depth of ablation generated by the 266 nm lasershots, samples that went through MSI were analyzed byconfocal fluorescence microscopy.

■ RESULTS AND DISCUSSION

All presented data are representative of six experiments.Determination of the Laser Shot Penetration Depth.

In mass spectrometry imaging, laser impacts may ablate theanalyzed sample, depending on the nature of the sample and onthe laser energy. Confocal fluorescence microscopy was used todetermine at which depth the laser interacts with the sampleduring MSI experiments. Figure 2 shows leaf surface imaged byfluorescence macroscopy (a) and confocal fluorescencemicroscopy (b−d) after MSI analysis. The settings of the 266nm laser were the same as for the other experiments describedin this article.Figure 2a shows that LDI produced blue-excited green

fluorescence in the abaxial epidermis of the leaf. More precisely,

this green fluorescence induced by the laser impacts duringMSI was located in epidermal cell walls (Figure 2b). Confocalfluorescence microscopy did not reveal any holes on the leafsurface (Figure 2b−d). Therefore, the desorption/ionization266 nm laser used for LDI-MSI did not dig into the samples. Itwas operated here under desorption conditions. Stilbenesdetected with MSI come only from the leaf surface, a fewmicrons in depth at most.

Stilbene Detection by Mass Spectrometry. Thisprotocol of successive imaging was first applied to grapevineleaves irradiated by UV−C. This abiotic stress leads to thebiosynthesis of stilbenes in high amounts over the wholeirradiated area. The synthesized stilbenes are mainly trans-resveratrol, trans-pterostilbene, cis- and trans-piceid, trans- andcis-δ-viniferin, and trans- and cis-ε-viniferin.46 The positive LDI-TOFMS mode coupled with a 266 nm laser leads to a verysensitive detection of molecular radical ions at m/z 228 and 256for resveratrol and pterostilbene, respectively.43 There isanother signal, higher than the one at m/z 256, forpterostilbene at m/z 254 corresponding to the [M − 2H]•+

ion. The formation of this species can be explained by a two-step photochemical process involving the conversion of amethoxylated stilbene compound into a phenanthrene species,as observed for the tetra-methoxylated stilbene.47 We firstinvestigated the detection of the major glycosylated resveratrolisomer, the trans-piceid under the present LDI conditions usingits relative standard (Figure 3).At least two signals attributed to trans-piceid were observed.

The first was related to the radical cation M•+ of trans-piceid.The second, much more intense, was detected at m/z 228. Thisproduct ion is detected following the loss of the glycosylatedmoiety (−162 u). The ether bond between the aglycon and theglucose breaks during the laser ionization/desorption process.The ionization yield of piceids is thus very low. Because of thegeneration of this product peaking at m/z 228, piceids willcontribute to the same m/z signal as the molecular ion of trans-resveratrol. The ionization of viniferins was also investigatedthrough the analysis of standards. No signal was detected underpresent LDI conditions. To achieve the ionization of thesecompounds, the help of a matrix (MALDI) is required. Forinstance, the 2,5-dihydroxybenzoic acid allows the ionization ofthe trans-δ-viniferin.44 However, investigating grapevine leavesby MSI without the need to apply a matrix layer isadvantageous, given the simplicity of sample preparation andthe avoidance of potential artifact generation.48 Because of allthe above, only m/z 228 and 254 ions were monitored by LDI-MSI.

Correlative Analysis of Stilbene in Leaves. In additionto the correlation between MSI and fluorescence imaging,stilbene response to both stresses (UV−C and infection by P.viticola) was analyzed by HPLC-DAD for all leaf samples.Trans-resveratrol, trans-pterostilbene, cis-piceid, trans-piceid,and viniferin contents of stressed and control leaf regionswere assessed. Indeed, all of these stilbenes contribute to thefluorescence signal under our experimental conditions,23 andLDI-MSI is sensitive to some of them. Table 1 presents theresults of the HPLC-DAD analysis for the UV−C treated leafand the leaf infected by P. viticola.As expected, the HPLC-DAD data confirm that both biotic

and abiotic stresses induce the synthesis of stilbenes. Overall,the increase of stilbene content was much higher for the leafexposed to UV−C than for the infected leaf: 5.33 mg/g DM forthe 180 s-irradiated area as opposed to 1.48 mg/g DM for the

Figure 2. (a) Fluorescence macroscopy and (b−d) confocalfluorescence microscopy images from the abaxial side of a leafpreviously analyzed by mass spectrometry imaging: the green part in(a). The right part of the leaf was used as control area: dark part in (a)and red structures in (b−d) due to chlorophyll fluorescence. (b) 2Dimage of the surface and (c, d) 3D projections of the same area (samescale for b−d). The orientation of the projections are indicated by x, y,and z signs in the figure: oblique in (c) and sagittal in (d).

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infected leaf at 4 dpi. However, viniferin contents werecomparable in both experiments. Unlike resveratrol, pteros-tilbene content was higher for the intermediate irradiated area(0.29 mg/g DM) than for the 180 s-irradiated area (0.09 mg/gDM), which was unexpected. Piceids showed the same trend,but less marked. This different behavior cannot yet beexplained. Infected leaves had more piceids than resveratrol.Piceids could therefore contribute significantly to the signal atm/z 228 (see part 3.2).Figure 4 shows pictures from the analysis performed on an

UV−C irradiated leaf. The color scale used for MSI mapsrepresents the relative intensity for each ion. The black color isused when no signal was detected in the corresponding pixel,whereas the white color represents the maximum intensity inthe map. This sample had three different zones, a control zonekept away from UV−C irradiations (on the right), anintermediate zone irradiated for 45 s (on the left), and azone irradiated for 180 s (in the middle). These different areascan be easily differentiated in MSI. Neither of the stilbenesignals (m/z 228 and 254) were detected in the control zone(Figure 4c,d). The resveratrol distribution allowed one todistinguish clearly the three zones. In the middle zone (Figure4c), resveratrol was uniformly distributed, whereas the 45 s-irradiation zone showed a few intense pixels (Figure 4c). Apartfrom these intense spots, the left zone of Figure 4c exhibited a

low content in resveratrol (dark blue pixels compared to blackpixels of the control zone). The MSI map of pterostilbeneshowed a different behavior from that of resveratrol. The MSIsignal of pterostilbene was higher in the left 45s-irradiated zonethan for the middle zone irradiated with UV−C during 180 s.HPLC-DAD confirmed this observation (see Table 1).Fluorescence imaging revealed stilbene signals only in theirradiated area (Figure 4b). The two areas irradiated withdifferent durations appear clearly on the RGB overlay (Figure4b). This is the consequence of two effects: (1) a larger andmore uniform fluorescence of stilbenes in the blue channel forthe middle 180 s-irradiated zone, because of the higher contentin stilbenes in this zone (Table 1); and (2) a completeextinction of the chlorophyll fluorescence in the red channeldue to the long UV−C treatment. This treatment damaged theleaves (photooxidation): brownish spots are numerous in themiddle zone (Figure 4a).The same protocol was then conducted on a leaf infected by

P. viticola in order to confirm the usefulness of this correlativeanalysis. A representative sample of the stilbene distributionobtained in this second type of experiment is shown in Figure5.In the infected area, the veins showed an intense blue

fluorescence in macroscopy (Figure 5b), whereas no vascularsignal was detected in MSI (Figure 5c,d). The same was true

Figure 3. LDI-TOFMS mass spectrum of a methanolic solution of a piceid standard (2 × 10−5 M) prepared as a 2 μL deposit. 100 laser shots wereused to record the mass spectrum. The fragmentation is specified on the molecular structure.

Table 1. Stilbene Quantification by HPLC-DAD of Methanolic Extracts (mg/g of Dry Matter) of the UV−C Irradiated LeafShown in Figure 4 and the P. viticola Infected Leaf Shown on Figure 5a

stilbene content (mg/g DM)

experiment treatment resveratrol pterostilbene piceids viniferins total content

UV−C irradiation control (0 s) N.D. N.D. 0.10 ± 0.01a N.D. 0.10 ± 0.01a

45 s 0.71 ± 0.03a 0.287 ± 0.016a 1.38 ± 0.10b 0.85 ± 0.09a 3.22 ± 0.34b

180 s 3.16 ± 0.12b 0.085 ± 0.005b 1.09 ± 0.08c 0.99 ± 0.11a 5.33 ± 0.57c

P. viticola infection control 0.070 ± 0.003a N.D. 0.16 ± 0.01a N.D. 0.23 ± 0.02a

infected 0.112 ± 0.004b 0.063 ± 0.004 0.70 ± 0.05b 0.61 ± 0.06 1.48 ± 0.16b

aData are mean ± SD, n = 6. Means followed by the same letters for a same column and a same experiment indicates that there is no significantdifference between them (p < 0.05). N.D.: not detected.

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Figure 4. Analysis of the UV−C irradiated grapevine leaf. (a) Transmission macroscopy and (b) fluorescence macroscopy RGB overlay; (c)resveratrol and piceids (m/z 228) and (d) pterostilbene (m/z 254) molecular maps. Frames indicate the common area analyzed with bothtechniques. Vertical lines mark out the 3 zones: the left zone, irradiated for 45 s; the middle zone, irradiated for 180 s; the right zone, not irradiated.Color scales for MSI images are expressed in relative intensity.

Figure 5. Analysis of an inoculated grapevine leaf: (a) transmission macroscopy and (b) fluorescence macroscopy RGB overlay; (c) resveratrol andpiceids (m/z 228) and (d) pterostilbene (m/z 254) molecular maps. Frames indicate the common area analyzed with both techniques. Thehorizontal line indicates the separation between the upper zone (infected) and the lower zone (control). Color scales for MSI images are expressedin relative intensity.

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for the control area. Veins fluoresce blue mainly due tohydroxycinnamic acids.49,50 By contrast, blue fluorescence andion distributions (MSI) were colocalized in the intercostalregions (areoles) of the infected area. In addition, the spatialdistribution was heterogeneous both in fluorescence imagingand MSI. Some spots were more intense and probablycorrespond to guard cells through which the infectionoccurs.23,24 Indeed, as shown by Poutaraud et al.,23 theheterogeneity in fluorescence microscopy images of stilbeneVBF in the intercostal regions is due to the higher bluefluorescence of stomata guard cells and cell walls. Fluorescenceyield of stilbene molecules increases with the rigidity of theirenvironment24 and guard cells and other lignified tissues aremore rigid than the other parts of the areoles. Therefore, it isnot possible to distinguish between two interpretations: thatthe higher stilbene VBF of guard cells and cell walls is due to ahigher content in stilbene or whether this is just due tomicroenvironment effects upon fluorescence yield.The resolution of fluorescence imaging (macroscopy) used

here was too low to distinguish cell walls. However, stomataguard cells, even if not properly resolved, could be inferred. Thegranularity of the MSI images with brighter pixels distributedrandomly, but regularly spaced, would indicate a higher contentin stilbene of stomata guard cells. Unfortunately, the resolutionof MSI is too low to identify stomata and guard cells. Thecorrelative imaging by MSI and fluorescence microscopy withan appropriate experimental design for a perfect superpositionof the two images would alleviate this problem of spatialresolution. It will combine the advantages of the two methods:the high resolution of fluorescence microscopy (to resolve andlocalize stomata) and the identification and relative quantifica-tion capacity of MSI. The overall MSI signal of stilbene waslower here than for the UV−C experiment (Figure 4), inaccordance with the stilbene content determined by HPLC-DAD (Table 1). The m/z 228 (resveratrol and piceids) and254 (pterostilbene) ions were colocalized on the upper leaf areainfected by P. viticola. The pterostilbene signal was lower thanthe resveratrol and piceid signals, which is again consistent withHPLC-DAD analysis (see Table 1). As the resveratrol contentwas low in the HPLC-DAD measurements, the m/z 228 signalmay come in large part from piceids.

■ CONCLUSIONS

A sampling protocol was successfully developed to investigatein situ the same grapevine leaf by two complementary imagingtechniques: fluorescence imaging and mass spectroscopyimaging. This procedure enabled to localize global stilbenefluorescence in UV−C irradiated or P. viticola infectedgrapevine leaves with high resolution and to observe trans-resveratrol, trans-pterostilbene, and piceids individual distribu-tions at a lower resolution. This correlative imaging approachcan contribute to understanding how the grapevine leaf defendsagainst environmental stresses. It confirmed here that grapevineleaves react differently in response to abiotic and biotic stress.There was a rather uniform synthesis of stilbenes (includingveins) induced by UV−C, whereas a rather localized synthesisof stilbenes in stomata guard cells and cell walls was induced byP. viticola infection. After this first demonstration of technicalfeasibility and of usefulness, this correlative analysis wouldgreatly benefit from the MALDI to allow localizing individuallyall main stilbene compounds synthesized by grapevine leaves.This approach could be extended to other pathosystems

involving fluorescent phytoalexins found in other species, suchas coumarins in sunflower or isoflavonoids in soybean.51

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: loic.becker@univ-lorraine.fr. Phone: (+33) 3 87 54 7068.*E-mail: vincent.carre@univ-lorraine.fr. Phone: (+33) 3 72 7491 33.ORCIDLoïc Becker: 0000-0002-1945-6564Anne Poutaraud: 0000-0002-1496-3196NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors acknowledge the financial support provided by the“Conseil Interprofessionnel du Vin de Bordeaux” (CIVB,Bordeaux, France). This work benefitted from the core facilitiesof Imagerie-Gif (http://www.i2bc.paris-saclay.fr), member ofIBiSA (http://www.ibisa.net), supported by “France-BioImag-ing” (ANR-10-ISBN-04-01), and the Labex “Saclay PlantScience” (ANR-11-IDEX-0003-02). We thank Jordi Molgo and Evelyne Benoit for generous access to their Zeiss confocalmicroscope at the Institut Federatif de Neurobiologie AlfredFessard.

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