Determination and imaging of metabolites from Vitis vinifera leaves by laser desorption/ionisation time-of- flight mass spectrometry Gregory Hamm 1 , Vincent Carre ´ 1 * , Anne Poutaraud 2 , Benoı ˆt Maunit 1 , Gilles Frache 1 , Didier Merdinoglu 2 and Jean-Franc ¸ois Muller 1 * 1 Laboratoire de Spectrome ´trie de Masse et de Chimie Laser, Institut Jean Barriol – Fe ´de ´ration de Recherche 2843, Universite ´ Paul Verlaine – Metz, 1 Boulevard Arago, 57078 Metz technopole Cedex 03, France 2 INRA, Laboratoire de ge ´ne ´tique et d’ame ´lioration de la vigne, UMR1131, 28 rue de Herrlisheim, B.P. 20507, 68021 Colmar Cedex, France Received 8 September 2009; Revised 20 November 2009; Accepted 20 November 2009 Analysis of grapevine phytoalexins at the surface of Vitis vinifera leaves has been achieved by laser desorption/ionisation time-of-flight mass spectrometry (LDI-ToFMS) without matrix deposition. This simple and rapid sampling method was successfully applied to map small organic compounds at the surface of grapevine leaves. It was also demonstrated that the laser wavelength is a highly critical parameter. Both 266 and 337 nm laser wavelengths were used but the 266 nm wavelength gave increased spatial resolution and better sensitivity for the detection of the targeted metabolites (resveratrol and linked stilbene compounds). Mass spectrometry imaging of grapevine Cabernet Sauvignon leaves revealed specific locations with respect to Plasmopara viticola pathogen infection or light illumination. Copyright # 2010 John Wiley & Sons, Ltd. In response to biotic or abiotic stresses, grapevines – like other plants – synthesise defense compounds. 1 Stilbenes are one of the secondary metabolites, also known as phytoalex- ines, involved in plant defense mechanisms. The relationship between plant stilbene content and grapevine disease resistance was first highlighted by Langcake. 2 Stilbenes have been recognised to prevent the proliferation and the reproduction of pathogens 3 such as downy mildew (Plasmopara viticola) on which numerous studies have been performed, 4,5 a major grapevine disease likely to cause the loss of much of the crop. When downy mildew affects the leaves of grapevines, oily lesions are observed on the upper surface while the under side is covered with a white cottony layer. Leaf death occurs shortly after and leads to the general weakening of the plant. The major stilbenes (Fig. 1) identified in infected grapevine are resveratrol (a) (3,5,4 0 -trihydroxy- stilbene) and its derivatives: pterostilbene (b) (3,5- dimethoxy-4 0 -hydroxystilbene), 6 piceid (resveratrol 3-O-b- D-glucoside), e-viniferin and d-viniferin (resveratrol dimers). Other factors may also induce stilbene synthesis such as UV irradiation, 7 aluminium chloride exposure, 8 or gray mold (Botritys cinerea) infection. 9 The study of plant/pathogen interactions requires tech- niques that allow the acquisition of data on micrometer scale of the characterised metabolites. Several techniques have been used to analyse stilbenes from organic solvent extracts of fresh leaves. These experiments require a preliminary separation step using chromatographic methods: high-performance liquid chromatography (HPLC) in normal or inverse phase 10 or gas chromatography with or without a derivatisation step. 11 Liquid chromatography techniques are easily coupled with a diode-array detector (DAD) to quantify stilbene and related compounds, and mass spectrometric analysers are used to provide elemental composition and structural data. To define the structures and stereochemistry of theses compounds, Huang et al. identified some novel tetramers of resveratrol in Vitis amurensis roots by two-dimensional nuclear magnetic resonance (2D NMR). 12 However, the leaf extracts studied by these techniques have generally been obtained from only a few square centimeters of tissue and thus cannot provide information on the precise localisation of secondary metab- olites on the leaf. This approach leads to a dilution of the local response induced by pathogens. Local assessment of stilbene contents on the surface of leaves may be also obtained by fluorescence spectroscopy, 13 as stilbenes display intense blue fluorescence under UV illumination. The fluorescence of the major stilbene content has been also correlated with HPLC-DAD measurements. Fluorescence spectroscopy allows the global stilbene fluor- escence per square millimeter on the leaf to be evaluated but does not achieve the separation of signals from different stilbenes. Imaging mass spectrometry (IMS) appears to be an interesting alternative to fluorescence mapping and liquid or gas chromatographic techniques. IMS allows a molecular map of all species to be achieved simultaneously. Further- more, this technique allows micrometer scale analysis to be obtained which is consistent with the size of stomata (20 mm), the site of infection by downy mildew. One of the first studies by imaging matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToFMS) was published by Caprioli et al. in RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2010; 24: 335–342 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4395 *Correspondence to: V. Carre ´, and J. Muller, Laboratoire de Spectrome ´trie de Masse et de Chimie Laser, Universite ´ Paul Verlaine – Metz, 1 Boulevard Arago, F-57078 Metz technopole Cedex 03, France. E-mail: carre@univ-metz.fr; jf.muller66@orange.fr Copyright # 2010 John Wiley & Sons, Ltd.