Correlative Analysis of Fluorescent Phytoalexins by Mass

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Correlative analysis of fluorescent phytoalexins by mass spectrometry imaging and fluorescence microscopy in grapevine leaves Loïc BECKER, Sébastien Bellow, Vincent Carré, Gwendal Latouche, Anne Poutaraud, Didier Merdinoglu, Spencer Brown, Zoran G. Cerovic, and Patrick CHAIMBAULT Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017 Downloaded from http://pubs.acs.org on June 1, 2017

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Analytical Chemistry

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Correlative analysis of fluorescent phytoalexins by

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mass

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microscopy in grapevine leaves

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Loïc Beckera*, Sébastien Bellowb, Vincent Carréa*, Gwendal Latoucheb, Anne Poutaraudc,d, Didier

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Merdinoglue,f, Spencer C. Browng, Zoran G. Cerovicb, Patrick Chaimbaulta

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a

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Complexes (LCP-A2MC), EA 4632, Institut Jean Barriol – Fédération de Recherche 2843; ICPM 1,

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Boulevard Arago ; Metz Technopole Cedex 03, F-57078, France.

9

b

spectrometry

imaging

and

fluorescence

Université de Lorraine. Laboratoire de Chimie et Physique-Approche Multi échelle des Milieux

Ecologie Systématique Evolution, Univ. Paris-Sud, CNRS, AgroParisTech, Université Paris-Saclay,

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91400, Orsay, France

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c

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Colmar Cedex, France.

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d

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de Haye - TSA 40602 - F54518 Vandœuvre-lès-Nancy Cedex, France.

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e

INRA, UMR 1131, SVQV,F-68000 Colmar, France

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f

Université de Strasbourg, UMR 1131, SVQV, F-68000 Colmar, France

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g

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Saclay, 91198, Gif‐sur‐Yvette cedex, France

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Corresponding authors:

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Loïc BECKER

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Unité de Recherche - Animal et Fonctionnalité des Produits Animaux

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Université de Lorraine

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1, Boulevard Arago

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F-57078 Metz Cedex 03 (France)

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[email protected]

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Phone (+33) 3 87 54 70 68

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Vincent CARRE

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Laboratoire de Chimie Physique - Approche Multi-Echelle des Milieux Complexes

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Université de Lorraine

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1, Boulevard Arago

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F-57078 Metz Cedex 03 (France)

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[email protected]

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Phone (+33) 3 72 74 91 33

INRA, Laboratoire Agronomie et Environnement, UMR 1121, Colmar, 29 rue de Herrlisheim, F68021

Université de Lorraine, Laboratoire Agronomie et Environnement, UMR 1121, 2 Avenue de la forêt

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris‐Sud, Université Paris‐

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ACS Paragon Plus Environment


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ABSTRACT

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Plant response to their environment stresses is a complex mechanism involving secondary

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metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a

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key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities,

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conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite

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localization during the extraction process. To overcome this issue, a correlative analysis combining

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mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ

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stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence

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provided by macroscopy were supplemented by specific distributions and structural information

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concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led

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to consistent and complementary data on the stilbene spatial distribution for the two stresses

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addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasis that grapevine

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leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced

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after UV-C irradiation whereas a more localized synthesis of stilbenes in stomata guard cells and cell

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walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to

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map phytoalexins of various plant tissues with resolution approaching the cellular level.

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Introduction

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Studies of secondary metabolites are a key to understand how plants respond to their environment,

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to stress and what mechanisms are involved. Stilbenes are phytoalexins produced in the

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phenylpropanoid pathway and are synthetized under biotic stress.1–3 The downy mildew disease,

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caused by the oomycete Plasmopara viticola, is one of these whose effects are well described.4

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Identified as phytoalexins of grapevine for the first time by Langcake and Pryce,5 stilbenes are known

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for their antifungal activity.6–10 Still, their effect on P. viticola mycelia remains a matter of debate.6

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Therefore, study of stilbenes in vivo would contribute to the understanding of the host-pathogen

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relationship. After P. viticola infection, grapevine leaves synthetize trans-resveratrol (3,5,4’-

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trihydroxystilbene), trans-pterostilbene (3,5 dimethoxy-4’-hydroxystilbene), trans- and -cis piceid (3-

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O-β-D-glucoside of resveratrol), and cyclic dehydrodimers of resveratrol trans-ε-viniferin and trans-δ-

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viniferin.11–16 Moreover, stilbenes can also be synthetized after abiotic stress such as UV-C

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irradiation,17 wound, dryness or chemicals.18 For their investigation, analytical techniques such as gas

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or liquid chromatography with UV and/or mass spectrometry detectors are traditionally used on a

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plant tissue extract.19,20 With these approaches, high levels of resolution and sensitivity are

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reached,21 but compound locations in tissues are lost. Indeed, prior to the analysis, these techniques

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require solvent extraction of the sample. This procedure homogenizes the molecular content of the

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sample. The mechanisms regulating stilbene synthesis appear complex;6 moreover, their location

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during the interaction with a pathogen or another stress is critical.

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Imaging techniques may provide metabolite distribution on the sample surface. Fluorescence

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imaging of stilbenes in grapevine leaves is based on their autofluorescence under UV light, as for

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several other phenolic compounds.22 Their violet-blue fluorescence (VBF) emission is centered

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around 390 nm both in methanol and in leaves. The maximum of excitation is around 320 nm.23

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Although a difference in fluorescence yield exists among the stilbenes produced by grapevine leaves,

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their fluorescence spectra are too similar to be used for discrimination in vivo.23,24 Stilbene 3



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localization has been studied by confocal fluorescence microscopy on leaves of grapevine genotypes

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with different levels of resistance to P. viticola.24 This technique enables in vivo visualization of

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phenolic compounds inside leaves by 3D reconstructions and optical sections.25 Moreover,

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fluorescence microscopy is nondestructive. Indeed, fluorescence imaging allows the observation of

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stilbenes in vivo,23,24 enabling kinetic studies on attached leaves. High resolution images of grapevine

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leaves have been obtained but without distinction between the different stilbenes.23,24,26

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Through mass spectrometry imaging (MSI), in situ compound identifications can be obtained at the

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same time as their surface area distributions. MSI dealing with plant metabolites has clearly

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emerged.27–31 Several metabolite families have been observed, such as agrochemicals in soya,32

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carbohydrates in wheat stems33 or wheat seeds,34 amino acids and phosphorylated molecules in

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wheat seeds,35 or even lipids present in an Asian variety of rice resistant to drought.36 Goto-Inoue et

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al. observed the location of the gamma-aminobutyric acid in eggplants.37 Other recent MSI studies of

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plant tissue deal with toxic glycoalkaloids in potato tuber,38 symbiosis of plants with nitrogen fixing

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microorganisms,39 anthocyanins in rice pericarp,40 and glucosinolates in Arabidopsis flowers and

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siliques.41 Parallel analyses in mass spectrometry were performed on grape berries by Berisha et al.

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using laser desorption followed by electrospray ionization (LD-ESI), MALDI imaging and HPLC/ESI-

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MS.42 This combined approach led to the localization of specific metabolites on the berry surface in

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addition to the characterization of several anthocyanins, amino acids and carbohydrates. Our

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previous reports of stilbene imaging were performed on grapevine leaves with laser

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desorption/ionization (LDI)43 and matrix assisted laser desorption/ionization (MALDI).44

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The present study assesses the feasibility to map plant metabolites combining MSI and fluorescence

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imaging. Laser desorption/ionization mass spectrometry imaging (LDI-MSI) allows characterizing and

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localizing stilbenes. However, MSI suffers from low spatial resolution, depending upon the laser

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features. The best spatial resolutions attained have ranged from 5 µm to 20 µm.45 Moreover, the LDI

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is a tough destructive process, which may result in high specificity of the ionized compounds. On the 4


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other hand, fluorescence imaging produces high-resolution images without damaging the sample.

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However, these two imaging techniques do not provide quantitative data in absolute values. The

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good correlation between VBF of stilbenes and total stilbene content shown by Poutaraud et al.23 is

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only valid at the macroscopic scale. In microscopic images, the intensity of stilbenes’ VBF cannot be

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used as a direct correlation to quantify stilbenes because of the major influence of the rigidity of

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stilbene molecules’ environment over their fluorescence yield and the large differences in the rigidity

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of the various tissue compartments.24 MSI provides molecular maps for each ion detected with semi-

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quantitative data in relative values. Indeed, the intensity of the pixel is proportional to the number of

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molecules of stilbenes from which the ion is issued. The relative value scales are thus specific to each

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ion and to each sample. To overcome this, HPLC coupled to a diode array detector (DAD) was used to

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add quantitative data in absolute values and to validate the compound identification of stilbenes. To

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test this approach, stressed grapevine leaves were studied. A first experiment was carried out on

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grapevine leaves treated by UV-C, which provokes stilbene synthesis on the whole treated surface.43

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A second experiment was then conducted on leaves infected by downy mildew.

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Experimental section Reagents

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Standard compounds of trans-resveratrol, trans-pterostilbene, trans-piceid, δ- and ε-viniferin and

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poly(-ethylene glycol) (PEG 600) were purchased from Sigma-Aldrich (Saint Quentin Fallavier,

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France). Trans-piceid, δ-viniferin and ε-viniferin were prepared in methanol at a concentration of 10-4

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M. For the LDI-ToFMS analysis of standards, 2 µL of each stilbene solution was deposited on the

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target.

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Plant material

5



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Hybrid genotypes of grapevines susceptible to P. viticola were studied. These hybrids resulted from

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crossings of the American species Muscadinia rotundifolia with Vitis vinifera cultivars. Plants were

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grown from green cuttings in Colmar (France) at 22 ± 3 °C with 13/11 light/dark in the greenhouse.

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The sixth leaf, counted from the apex of 3.5-month-old plants having 12–14 fully expanded leaves,

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was sampled and washed with demineralized water.

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Leaf UV-C irradiation

b)

a)

Infected 6 mm

i

ii

iii

6 mm

Control

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8 mm

8 mm

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Figure 1: protocols for (a) UV-C irradiation, (i) 45 s irradiated, (ii) 180 s irradiated, (iii) control area (non-irradiated), and (b) P. viticola infection of grapevine leaves. Three dots were deposited on each leaf with a felt-tip marker. They are used as reference marks to indicate the area to analyze by fluorescence microscopy and by mass spectrometry imaging.

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The abaxial side of leaves was exposed to UV-C radiations at 254 nm (UV-C tube, Osram, 30W,

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90 µWcm-2), at 13 cm distance from the lamp. The following protocol was applied to generate three

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different zones on the same sample: control (not irradiated), irradiated for 45 s, and irradiated for

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180 s. Two covers (i and iii) separated by 2 mm (ii) were positioned on the leaf (figure 1a). After 135 s

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of UV-C irradiation, the left cover (i) was removed. Then the leaf was further irradiated for 45 s. The

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middle area between the two covers (ii) was thus irradiated for 180 s. To allow the biochemical

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response to develop, the treated leaf was then maintained for three days in a closed petri dish with

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its adaxial side pressed against wet paper before fluorescence imaging.

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Leaf infection by P. viticola

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P. viticola was obtained from naturally infected plants in Colmar (France). Sporangia were

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periodically grown in order to prepare inoculants. The leaf was infected by spraying an inoculum 6


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solution at a concentration of 3.105 sporangia/mL on the upper half of the abaxial side of the leaf.

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The lower half of the leaf was protected with a cover, as described in figure 1b. During and after

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spraying, the leaves were put in 14-cm diameter petri dishes with the adaxial side pressed against

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wet paper. The petri dishes were closed just after spraying to maintain the leaves under moist

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conditions to favor inoculation and sporulation. Analyses were done after 4 days of incubation (4

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dpi).

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Fluorescence imaging (macroscopy)

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Images were acquired using a macroscope (AZ100 Multizoom, Nikon, Champigny-sur-Marne, France)

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equipped with a 130 W metal halide lamp white source (Intensilight, Nikon) and a high-resolution

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color camera (Ds-Ri, Nikon) at room temperature (19 °C). Macroscopy, as opposed to microscopy, is

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characterized by large object fields and large working distances, plus panning and zooming, allowing

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fluorescence imaging at organ, tissue and multicellular levels. The UV-suppression filter of this source

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was removed. The images of UV-excited visible autofluorescence were recorded using a custom-

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made filter block from AHF (Tübingen, Germany) with an excitation bandpass filter 340/26 (FF01

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Brightline, Semrock, Rochester, NY, USA), a dichroic filter Q380LP (Chroma Technology Corp., Bellows

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Falls, VT, USA), and a long-pass 371 nm emission filter (LP02-364RS, Semrock). The images of blue-

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excited green autofluorescence were recorded using a GFP-B filter set (excitation band pass filter

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472/30, dichroic filter 495 nm, and emission bandpass filter 520/35, Nikon). A ×2 objective (NA 0.2,

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working distanced 45 mm, AZ-Plan Fluor, Nikon) was used, and 24-bit RGB color images were

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acquired with a 1284 × 1024 pixel resolution. Imaged leaf pieces were flattened (abaxial side facing

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the objective) on the glass sample holder (adaxial side lightly moistened for adhesion). The flatness

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of the imaged area was necessary for a good-quality acquisition. When present, sporangiophores

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were washed from the sporulating leaves to avoid their contribution to VBF. Image acquisition was

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performed using the NIS-Elements software (Nikon). Image analysis, including composition, was 7



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performed using the software ImageJ (http://rsbweb.nih.gov/ij/). For the images of specific blue-

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excited green autofluorescence, only the green channel of the RGB pictures acquired was used,

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visualized with a black and green intensity scale (namely, look-up table - LUT). For the images of

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overall RGB UV-excited visible autofluorescence, images were processed by optimizing the brightness

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and contrast in each of the three color channels before making RGB overlays. This was necessary for

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a good and simultaneous visualization of both the chlorophyll fluorescence (red channel) and the

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blue fluorescence (blue channel and slightly green channel) that includes stilbene VBF.

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Confocal fluorescence microscopy and 3D image reconstruction

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The confocal microscope (LSM510 Meta, Zeiss, Jena, Germany) had an argon laser providing a

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488 nm beam dynamically filtered by an acousto-optic tunable filter (AOTF) that was used to excite

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the grapevine-leaf green autofluorescence. All experiments were performed with a x63 objective

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(Plan-Apochromat, NA 1.40 oil, Zeiss) at room temperature (19 °C). The dichroic filter used was HFT

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UV/488(Zeiss). Leaf samples were mounted in oil for microscopy (Immersol 518N, Zeiss) with the

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abaxial side facing the objective. The cover slips thickness was 0.170 mm (#1.5).

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The array detector of the Zeiss LSM510 Meta is a spectrograph dispersing emitted fluorescence from

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361.8 nm to 704.2 nm on a 32 photo-multiplier tube (PMT) array. The 32 signals were selectively

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binned for standard imaging. The images presented in this paper are the overlay of two detection

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channels on the Meta array detector: 500.9–597.2 nm (green channel), and 629.3–682.8 nm (red

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channel). Series of XY images, called Z-stack, were acquired along the Z axis, the axis perpendicular to

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leaf surface and parallel to the excitation beam. The optimal voxel size of 0.26 x 0.26 x 0.63 µm for x,

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y and z directions, respectively, was used. The resolution of the acquired images was 512 x 512

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pixels, coded in 8 bits for each color channel. The Z-stacks allowed a 3D analysis that is shown here

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through 3D projections. Image acquisition was performed using the software Zen (Zeiss). Image

8


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analysis, including 3D reconstruction, was carried out using the software LSM Image Browser (Zeiss)

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and the software ImageJ (http://rsbweb.nih.gov/ij/).

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Mass spectrometry imaging

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A Bruker Reflex IV MALDI-ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) was used to

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perform imaging experiments and to analyze standards, at room temperature (19 °C). In addition to

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the original nitrogen laser (337 nm, Science Inc., Boston, MA, USA), a second optical pathway into the

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ionization chamber was developed in our laboratory, that enabled us to perform LDI-MS experiments

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at 266 nm by coupling a quadrupled Nd-YAG laser (Continuum, Santa Clara, CA, USA). Positive mass

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spectra were acquired in the m/z 0-1000 range. The mass spectrometer was operated in the

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reflectron mode at a total acceleration voltage of 20 kV and a reflecting voltage of 23 kV. A delay

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time of 200 ns was used prior to ion extraction. The laser energy was kept at 60 % of its maximum

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value (fluence ≈ 0.5 J/cm2). The laser had a pulse duration of 5 ns and was used at a repetition rate of

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9 Hz. Mass spectra obtained for each pixel corresponded to the averaged mass spectrum of 50

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consecutive laser shots on the same location. The laser spot diameter was measured at 45 µm,

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therefore, spatial resolution was fixed at 50 µm. FlexImaging software (v.2.1, Bruker Daltonics,

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Bremen, Germany) was used to perform mass spectrometry imaging experiments. PEG 600 (10-2 M)

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was used to perform external calibration. Approximately 12 h were required to achieve an image of

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about 7000 pixels.

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Methanolic extraction and HPLC-DAD analysis

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The extraction protocol was derived from the method used by Pezet et al.14 Foliar discs of 2 cm

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diameter were collected close to the imaged area for each condition: control (neither irradiated nor

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inoculated), irradiated for 45 s, irradiated for 180 s and infected areas. These were placed in 1 mL of

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methanol for extraction. A ratio of dry matter to solvent volume less than 15 mg/mL was maintained 9



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for each extraction. Samples were then placed in a water bath at 60 °C for 45 min with stirring. The

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extracts were centrifuged and stored at -20 °C before HPLC analysis. Stilbene quantification was

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performed with a 1100 HPLC system (Hewlett-Packard, Agilent Technologies, Massy, France)

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equipped with a diode array detector (Hewlett-Packard, 190 nm to 950 nm). Separation was carried

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on a RP-18 "end capped" 5 µm 130 Å column (LiChrospher, Merck, Lyon, France) of 250-mm length

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and 4.6 mm inner diameter, thermostatted at 20 °C. The solvent system was the one described by

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Pezet et al.14. Chromatograms were recorded at 307 nm. Contents are expressed as means of six

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replicates ± standard deviations. Statistical analysis was conducted using R software (v3.2.5, R Core

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Team). Mean values were compared by using Student’s t test at p < 0.05 significance level.

230 231

Handling for correlative analyses

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Samples were imaged first by fluorescence and second by LDI-MSI. Immediately after fluorescence

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imaging, samples were freeze-dried between two microscope glass slides covered by tape to avoid

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damage (warping and cracking). This fixed the sample state, so we could observe the same sample

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with both imaging techniques. Three corners were marked with a felt-tip pen to define the zone

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selected for imaging (figure 1). Molecular maps were processed and extracted with the FlexImaging

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software. To determine the depth of ablation generated by the 266 nm laser shots, samples that

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went through MSI were analyzed by confocal fluorescence microscopy.

239 240

Results and discussion

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All presented data are representative of six experiments.

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Determination of the laser shot penetration depth

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In mass spectrometry imaging, laser impacts may ablate the analyzed sample, depending on the

245

nature of the sample and on the laser energy. Confocal fluorescence microscopy was used to 10


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determine at which depth the laser interacts with the sample during MSI experiments. Figure 2

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shows leaf surface imaged by fluorescence macroscopy (a) and confocal fluorescence microscopy (b,

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c and d) after MSI analysis. The settings of the 266 nm laser were the same as for the other

249

experiments described in this article.

250 251 252 253 254 255

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

256

Figure 2a) shows that LDI produced blue-excited green fluorescence in the abaxial epidermis of the

257

leaf. More precisely, this green fluorescence induced by the laser impacts during MSI was located in

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epidermal cell walls (fig.2b). Confocal fluorescence microscopy did not reveal any holes on the leaf

259

surface (fig.2b, c, d). Therefore the desorption/ionization 266-nm laser used for LDI-MSI did not dig 11



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260

into the samples. It was operated here under desorption conditions. Stilbenes detected with MSI

261

come only from the leaf surface, a few microns in depth at most.

262 263

Stilbene detection by mass spectrometry

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This protocol of successive imaging was first applied to grapevine leaves irradiated by UV-C. This

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abiotic stress leads to the biosynthesis of stilbenes in high amounts over the whole irradiated area.

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The synthesized stilbenes are mainly trans-resveratrol, trans-pterostilbene, cis- and trans-piceid,

267

trans- and cis-δ-viniferin and trans- and cis-ε-viniferin.46 The positive LDI-TOFMS mode coupled with

268

a 266-nm laser leads to a very sensitive detection of molecular radical ions at m/z 228 and 256 for

269

resveratrol and pterostilbene, respectively.43 There is another signal, higher than the one at m/z 256,

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for pterostilbene at m/z 254 corresponding to the [M-2H]•+ ion. The formation of this species can be

271

explained by a two-step photochemical process involving the conversion of a methoxylated stilbene

272

compound into a phenanthrene species, as observed for the tetra-methoxylated stilbene.47 We first

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investigated the detection of the major glycosylated resveratrol isomer, the trans-piceid under the

274

present LDI conditions using its relative standard (figure 3).

275

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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 were used to record the mass spectrum. The fragmentation is specified on the molecular structure.

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At least two signals attributed to trans-piceid were observed. The first was related to the radical

279

cation M•+ of trans-piceid. The second, much more intense, was detected at m/z 228. This product

280

ion is detected following the loss of the glycosylated moiety (-162 u). The ether bond between the

281

aglycon and the glucose breaks during the laser ionization/desorption process. The ionization yield of

282

piceids is thus very low. Because of the generation of this product peaking at m/z 228, piceids will

283

contribute to the same m/z signal as molecular ion of trans-resveratrol. The ionization of viniferins

284

was also investigated through the analysis of standards. No signal was detected under present LDI

285

conditions. To achieve the ionization of these compounds, the help of a matrix (MALDI) is required.

286

For instance, the 2.5-dihydroxybenzoic acid allows the ionization of the trans-δ-viniferin.44 However,

287

investigating grapevine leaves by MSI without the need to apply a matrix layer is advantageous,

288

given the simplicity of sample preparation and the avoidance of potential artifact generation.48

289

Because of all the above, only m/z 228 and 254 ions were monitored by LDI-MSI.

290 291

Correlative analysis of stilbene in leaves

292

In addition to the correlation between MSI and fluorescence imaging, stilbene response to both

293

stresses (UV-C and infection by P. viticola) was analyzed by HPLC-DAD for all leaf samples. Trans-

294

resveratrol, trans-pterostilbene, cis-piceids, trans-piceid, and viniferin contents of stressed and

295

control leaf regions were assessed. Indeed, all of these stilbenes contribute to the fluorescence signal

296

under our experimental conditions23 and LDI-MSI is sensitive to some of them. Table 1 presents the

297

results of the HPLC-DAD analysis for the UV-C treated leaf and the leaf infected by P. viticola.

298 299

Table 1: stilbene quantification by HPLC-DAD of methanolic extracts (mg/g of dry matter) of the UV-C irradiated leaf shown on figure 4 and the P. viticola infected leaf shown on figure 5. "N.D." not detected.

Experiment UV-C irradiation P. viticola

Treatment Control (0 sec) 45 sec 180 sec control

Stilbene content (mg/g DM) Resveratrol Pterostilbene Piceids Viniferins N.D. N.D. a a 0.71 ± 0.03 0.287 ± 0.016 b b 3.16 ± 0.12 0.085 ± 0.005 a 0.070 ± 0.003 N.D.

a

0.10 ± 0.01 b 1.38 ± 0.10 c 1.09 ± 0.08 a 0.16 ± 0.01

N.D. a 0.85 ± 0.09 a 0.99 ± 0.11 N.D.

13


Total content a

0.10 ± 0.01 b 3.22 ± 0.34 c 5.33 ± 0.57 a 0.23 ± 0.02


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

infection

b

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b

infected 0.112 ± 0.004 0.063 ± 0.004 0.70 ± 0.05 0.61 ± 0.06 1.48 ± 0.16 Data are mean ± SD, n = 6. Means followed by the same letters for a same column and a same experiment indicates that there is no significant difference between them (p < 0.05).

b

300 301

As expected, the HPLC-DAD data confirm that both biotic and abiotic stresses induce the synthesis of

302

stilbenes. Overall, the increase of stilbene content was much higher for the leaf exposed to UV-C

303

than for the infected leaf: 5.33 mg/g DM for the 180s-irradiated area as opposed to 1.48 mg/g DM

304

for the infected leaf at 4 dpi. However, viniferin contents were comparable in both experiments.

305

Unlike resveratrol, pterostilbene content was higher for the intermediate irradiated area (0.29 mg/g

306

DM) than for the 180s-irradiated area (0.09 mg/g DM) which was unexpected. Piceids showed the

307

same trend, but less marked. This different behavior cannot yet be explained. Infected leaves had

308

more piceids than resveratrol. Piceids could therefore contribute significantly to the signal at m/z 228

309

(see part 3.2).

310 14


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311 312 313 314

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 both techniques. 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.

315

Figure 4 shows pictures from the analysis performed on an UV-C irradiated leaf. The color scale used

316

for MSI maps represents the relative intensity for each ion. The black color is used when no signal

317

was detected in the corresponding pixel, whereas the white color represents the maximum intensity

318

in the map. This sample had three different zones, a control zone kept away from UV-C irradiations

319

(on the right), an intermediate zone irradiated for 45 s (on the left), and a zone irradiated for 180 s

320

(in the middle). These different areas can be easily differentiated in MSI. Neither of the stilbene

321

signals (m/z 228 and 254) were detected in the control zone (fig.4c, d). The resveratrol distribution

322

allowed one to distinguish clearly the three zones. In the middle zone (fig.4c) resveratrol was

323

uniformly distributed, whereas the 45s- irradiation zone showed a few intense pixels (fig.4c). Apart

324

from these intense spots, the left zone of fig.4c exhibited a low content in resveratrol (dark blue

325

pixels compared to black pixels of the control zone). The MSI map of pterostilbene showed a

326

different behavior from that of resveratrol. The MSI signal of pterostilbene was higher in the left 45s-

327

irradiated zone than for the middle zone irradiated with UV-C during 180 s. HPLC-DAD confirmed this

328

observation (see table 1). Fluorescence imaging revealed stilbene signals only in the irradiated area

329

(fig.4b). The two areas irradiated with different durations appear clearly on the RGB overlay (fig.4b).

330

This is the consequence of two effects: 1) a larger and more uniform fluorescence of stilbenes in the

331

blue channel for the middle 180s-irradiated zone, because of the higher content in stilbenes in this

332

zone (table 1); and 2) a complete extinction of the chlorophyll fluorescence in the red channel due to

333

the long UV-C treatment. This treatment damaged the leaves (photooxidation): brownish spots are

334

numerous in the middle zone (fig.4a).

335

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

336

The same protocol was then conducted on a leaf infected by P. viticola in order to confirm the

337

usefulness of this correlative analysis. A representative sample of the stilbene distribution obtained

338

in this second type of experiment is showed in figure 5.

339 340 341 342 343

Figure 5: analysis of an inoculated 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 both techniques. The horizontal line indicates the separation between the upper zone (infected) and the lower zone (control). Color scales for MSI images are expressed in relative intensity.

344

In the infected area, the veins showed an intense blue fluorescence in macroscopy (fig.5b) whereas

345

no vascular signal was detected in MSI (fig.5c, d). The same was true for the control area. Veins

346

fluoresce blue mainly due to hydroxycinnamic acids.49,50 By contrast, blue fluorescence and ion

347

distributions (MSI) were colocalized in the intercostal regions (areoles) of the infected area. In

348

addition, the spatial distribution was heterogeneous both in fluorescence imaging and MSI. Some

349

spots were more intense and probably correspond to guard cells through which the infection

350

occurs.23,24 Indeed, as shown by Poutaraud et al.23, the heterogeneity in fluorescence microscopy 16


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351

images of stilbene VBF in the intercostal regions is due to the higher blue fluorescence of stomata

352

guard cells and cell walls. Fluorescence yield of stilbene molecules increases with the rigidity of their

353

environment24 and guard cells and other lignified tissues are more rigid than the other parts of the

354

areoles. Therefore, it is not possible to distinguish between two interpretations: that the higher

355

stilbene VBF of guard cells and cell walls is due to a higher content in stilbene or whether this is just

356

due to microenvironment effects upon fluorescence yield.

357 358

The resolution of fluorescence imaging (macroscopy) used here was too low to distinguish cell walls.

359

However, stomata guard cells, even if not properly resolved, could be inferred. The granularity of the

360

MSI images with brighter pixels distributed randomly, but regularly spaced, would indicate a higher

361

content in stilbene of stomata guard cells. Unfortunately, the resolution of MSI is too low to identify

362

stomata and guard cells. The correlative imaging by MSI and fluorescence microscopy with an

363

appropriate experimental design for a perfect superposition of the two images would alleviate this

364

problem of spatial resolution. It will combine the advantages of the two methods: the high resolution

365

of fluorescence microscopy (to resolve and localize stomata) and the identification and relative

366

quantification capacity of MSI. The overall MSI signal of stilbene was lower here than for the UV-C

367

experiment (fig. 4), in accordance with the stilbene content determined by HPLC-DAD (table 1). The

368

m/z 228 (resveratrol and piceids) and 254 (pterostilbene) ions were colocalized on the upper leaf

369

area infected by P. viticola. The pterostilbene signal was lower than the resveratrol and piceid

370

signals, which is again consistent with HPLC-DAD analysis (see table 1). As the resveratrol content

371

was low in the HPLC-DAD measurements, the m/z 228 signal may come in large part from piceids.

372 373

Conclusions

374

A sampling protocol was successfully developed to investigate in situ the same grapevine leaf by two

375

complementary imaging techniques: fluorescence imaging and mass spectroscopy imaging. This 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

376

procedure enabled to localize global stilbene fluorescence in UV-C irradiated or P. viticola infected

377

grapevine leaves with high resolution and to observe trans-resveratrol, trans-pterostilbene, and

378

piceids individual distributions at a lower resolution. This correlative imaging approach can

379

contribute to understanding how the grapevine leaf defends against environmental stresses. It

380

confirmed here that grapevine leaves react differently in response to abiotic and biotic stress. There

381

was a rather uniform synthesis of stilbenes (including veins) induced by UV-C whereas a rather

382

localized synthesis of stilbenes in stomata guard cells and cell walls was induced by P. viticola

383

infection. After this first demonstration of technical feasibility and of usefulness, this correlative

384

analysis would greatly benefit from the MALDI to allow localizing individually all main stilbene

385

compounds synthetized by grapevine leaves. This approach could be extended to other

386

pathosystems involving fluorescent phytoalexins found in other species, such as coumarins in

387

sunflower or isoflavonoids in soybean.51

388 389

Acknowledgements

390

The authors acknowledge the financial support provided by the "Conseil Interprofessionnel du Vin de

391

Bordeaux" (CIVB, Bordeaux, France). This work benefitted from the core facilities of Imagerie‐Gif,

392

(http://www.i2bc.paris‐saclay.fr), member of IBiSA (http://www.ibisa.net), supported by “France‐

393

BioImaging” (ANR‐10‐ISBN‐04‐01), and the Labex “Saclay Plant Science” (ANR‐11‐IDEX‐0003‐02). We

394

thank Jordi Molgó and Evelyne Benoît for generous access to their Zeiss confocal microscope at the

395

Institut Fédératif de Neurobiologie Alfred Fessard.

396 397

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480


For TOC only

481

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2: (a) fluorescence macroscopy and (b; c; d) confocal fluorescence microscopy images from the abaxial side of a leaf previously analyzed by mass spectrometry imaging – the green part (a). The right part of the leaf was used as control area – dark part in (a) and red structures in (b; c; d) due to chlorophyll fluorescence. (b) is a 2D image of the surface and (c; d) are 3D projections of the same area (same scale for b; c; d). The orientation of the projections are indicated by x, y, and z signs in the figure, oblique in (c) and sagittal in (d). 140x140mm (150 x 150 DPI)


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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 both techniques. 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. 179x145mm (150 x 150 DPI)



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Figure 5: analysis of an inoculated 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 both techniques. The horizontal line indicates the separation between the upper zone (infected) and the lower zone (control). Color scales for MSI images are expressed in relative intensity. 176x134mm (150 x 150 DPI)


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For TOC only 168x94mm (150 x 150 DPI)


Correlative Analysis of Fluorescent Phytoalexins by Mass

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