Determination and imaging of metabolites from Vitis vinifera leaves by laser desorption/ionisation...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2010; 24: 335–342

) DOI: 10.1002/rcm.4395
Published online in Wiley InterScience (www.interscience.wiley.com
Determination and imaging of metabolites from Vitisvinifera leaves by laser desorption/ionisation time-of-

flight mass spectrometry

Gregory Hamm1, Vincent Carre1*, Anne Poutaraud2, Benoıt Maunit1, Gilles Frache1,

Didier Merdinoglu2 and Jean-Francois Muller1*1Laboratoire de Spectrometrie de Masse et de Chimie Laser, Institut Jean Barriol – Federation de Recherche 2843, Universite Paul Verlaine –

Metz, 1 Boulevard Arago, 57078 Metz technopole Cedex 03, France2INRA, Laboratoire de genetique et d’amelioration 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

*CorrespoSpectromVerlaineCedex 03E-mail: c

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 337nm laser wavelengths were used but the 266nmwavelength 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 pathogens3 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) (3,5,40-trihydroxy-

stilbene) and its derivatives: pterostilbene (b) (3,5-

dimethoxy-40-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

ndence to: V. Carre, and J. Muller, Laboratoire deetrie de Masse et de Chimie Laser, Universite Paul– Metz, 1 Boulevard Arago, F-57078 Metz technopole, [email protected]; [email protected]

liquid chromatography (HPLC) in normal or inverse phase10

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

(�20mm), 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

Copyright # 2010 John Wiley & Sons, Ltd.

Figure 1. Chemical structures of trans-resveratrol (a) and

trans-pterostilbene (b).

336 G. Hamm et al.

1997.14 Caprioli’s group has carried out MALDI imaging of

proteins and peptides from thin sections of human and rat

tissues. Recently, profiling of amino acids, sugars and

phosphorylated compounds from wheat seeds15 or fatty

acids and flavonoids from fruits16 has been investigated.

However, to our best knowledge, there have been no reports

of the detection and imaging of grapevine phytoalexins on

the surface of grapevine leaf using direct LDI-ToFMS. Since

the introduction of IMS, many development have been

carried out to improve the technique such as spatial

resolution with a microprobe approach,17,18 data treatment19

and software,20 or new matrices.16,21 Static secondary ion

mass spectrometry (SIMS)22 is another technique which

offers very high spatial resolution – at the tenth of nanometer

scale.23 However, the chemical information is limited to the

uppermost surface of a sample and it is generally difficult to

detect intact ions from moleculaes.24 In MALDI-MS imaging,

in spite of matrix deposition improvements,25,26 one major

difficulty is the inhomogeneous matrix deposition.

The aim of this work is to demonstrate that matrix-free

laser desorption/ionisation time-of-flight mass spectrometry

is a powerful tool for in situ studies of biological sample such

as grapevine leaves in order to detect and map small organic

compounds. LDI-ToFMS at 266 nm wavelength has been

used for ion imaging of resveratrol and pterostilbene on the

surface of Cabernet Sauvignon leaves. Cabernet Sauvignon

which is the most widespread variety in French vineyards is

also susceptible to downy mildew infection. The relative

concentration of stilbenes on the leaf has been assessed and

some correlation between the site of the infection or

irradiation and the synthesis of stilbene has been established.

EXPERIMENTAL

Plant material and leaf sample preparationPlants of Cabernet Sauvignon Vitis vinifera were propagated

in greenhouse from cuttings. The sixth leaf, counted from the

apex of 3.5-month-old plants having 12–14 fully expanded

leaves, was sampled and washed with demineralised water.

Leaves were divided into three different plots: (a) control

leaves, which were not infected; (b) Plasmopara viticola-

infected leaves by spraying a 105 sporangia.mL�1 solution of

Plasmopara viticola sporangia on the abaxial side; and (c)

irradiated leaves by a UV-C lamp at the 254 nm wavelength

(Osram, Molsheim, France; 30 W, 90mW.cm�2) for 5 min. The

distance between lamp and a leaf was kept to 13 cm.

Five days after treatments (b) and (c), discs were cut out

from each leaf using a 4-mm-diameter hollow-punch and

fixed onto a metal MALDI target plate with aluminised tape.

No MALDI matrix was added. Leaf discs were placed in high


vacuum to stop phytoalexin synthesis and stored at room

temperature before imaging experiments.

Standard and solventsStandard resveratrol and pterostilbene were purchased from

Sigma-Aldrich (Munchen, Germany). To avoid any pollution

HPLC-grade solvents were tri-distilled before use. Poly(-

ethylene glycol) (PEG 600) (Fluka, Buchs, Switzerland) was

used to achieve calibration. For standard LDI-ToFMS

analysis, 1mL of a resveratrol and pterostilbene methanolic

solution at concentrations of 10�4 M and 10�2 M, respect-

ively, was deposited on the target. PEG 600 solution

(2mL; 10�2 M) was also added directly to the leaf surface

before the in situ experiment.

Mass spectrometry analysisA Bruker Reflex IV MALDI-ToF mass spectrometer (Bruker

Daltonics, Bremen, Germany) was used to perform in situ

analysis and imaging experiments. In addition to the original

nitrogen laser (l¼ 337 nm, Science Inc., Boston, MA, USA), a

second optical pathway into the ionisation chamber was

developed in our laboratory,27 which enabled to us perform

LDI-MS experiments at 266 nm wavelength by coupling a

quadrupled Nd-YAG laser (Continuum, Santa Clara, CA,

USA). Positive mass spectra were acquired in the m/z 0–1000

range. The mass spectrometer was operated in the reflectron

mode at a total acceleration voltage of 20 kV and a reflecting

voltage of 23 kV. A delay time of 200 ns was used prior to ion

extraction. The laser energy was kept at 65% of its maximum

value (fluence �0.5 J/cm2). The laser (pulse duration 5 ns)

was used at a repetition rate of 8 Hz. The mass spectrum

obtained for each pixel of the images corresponds to the

averaged mass spectrum of 50 consecutive laser shots on the

same location. Approximately 5 h were required to achieve a

0.9� 1.8 mm image with a 25mm spatial resolution (total

pixel number 2592). LDI-Fourier transform ion cyclotron

resonance mass spectrometric (FTICRMS) measurements

were conducted on an Explorer HiRes FTICR mass

spectrometer (Varian Corporation, Palo Alto, CA, USA)

fitted with a 9.4 T shielded superconducting magnet and a

ProMALDI ionisation source at 355 nm obtained from a

tripled Nd-YAG laser.

Imaging method and softwareDifferent software and steps are required to perform

experiments with our IMS device. First, a virtual grid of the

investigated sample has to be generated by using the Create a

target software.20 This allows the size, the inter-pixel distance,

the number of pixels and the location of sample on MALDI

target plate to be defined. The resulting file is then imported

into Flex Control (Bruker-Daltonics), which ensures control of

the mass spectrometer. The Autoexecute function allows mass

spectra to be automatically acquired. An image file is finally

generated by conversion of the mass spectrum obtained for

each pixel and association with its X and Y coordinates by

means of the Analyze this! software.20 The visualisation of the

image is ensured by Biomap software (Novartis, Basel,

Switzerland). Additional information on the different software

and the general procedure have been well described by

Clerens et al.20 All pieces of software are freely available.28


DOI: 10.1002/rcm

Figure 2. Example of 200 mesh nickel grid imaging in

positive ion detection mode. (a) Optical image of imaging

region. (b) LDI-ToF image of m/z 57.935 corresponding to

the nickel 58Niþ ion.

Imaging of metabolites from Vitis vinifera leaves 337

Development of LDI-MS imagingThe critical parameter in LDI-MS especially in microprobe

LDI-IMS is the laser wavelength. Images and analyses were

mainly acquired in this work at 266 nm wavelength for two

main reasons. The first is the increased sensitivity for the

detection of the investigated metabolite compounds and the

second is the ability to significantly decrease the laser spot

diameter at this wavelength compared with what it may be

obtained at 337 nm with a nitrogen laser. This parameter has

to be carefully tuned to get reproducible images. Different

devices have to be added to the 266 nm laser pathway to

increase the homogeneity of the laser beam.27 These consist

mainly of high optical quality telescope, lenses and prisms. In

addition, a pinhole is used to select only the central

homogeneous part (Gaussian profile) of the laser beam.

Analysis of the laser impacts by optical microscopy allows

the quality of the laser matter interaction to be checked and

the size of the laser impact to be evaluated. As previously

reported by Spengler et al. the size of the laser impact was

measured after interaction of a laser shot with red ink placed

on the MALDI sample holder.29 The laser fluence used is

identical to that required to perform IMS on grapevine

leaves. A laser spot diameter in the 10mm range was typically

obtained.

The decrease in laser spot diameter also ensures an

improvement of the mass resolution without significant loss

of sensitivity. For example, a resolving power up to 15 000 for

the m/z 578.527 ion was achieved. A small size as well as a

more homogeneous laser beam explained this behavior as

the kinetic energy distribution is sharper than that obtained

with a N2 laser beam.30 Under these conditions, the typical

mass accuracy is better than 25 ppm in the m/z range of 20 to

1000. To ensure higher laser output energy stability over the

time required for the acquisition of an image, a 15 min warm-

up step was added.

To investigate the ability of the proposed protocol, an

image of a 200 mesh nickel microscopic grid fixed on the

MALDI sample holder was acquired by LDI-MS (Fig. 2). The

image was constructed after the acquisition of 40� 40 mass

spectra corresponding to a 0.8� 0.8 mm investigated area.

The image was built using the m/z 57.935 signal, which

corresponds to the most abundant nickel isotope. The

resulting image was compared with an optical image of

the same area obtained with a digital camera (CCD DXC-

107P, Sony, Tokyo, Japan) connected to a Wild M3Z

microscope (Wild Heerbrugg, Heerbrugg, Switzerland).

The same contrasts were observed with both imaging

techniques. The contours of the microscopic grid were

found to be clearly highlighted on the m/z 57.935 mass

spectrometry image. In the figure the color varies from black

to white with respect to the 58Niþ ion signal. Mass spectra

were acquired from left to right and from top to bottom. The

decrease in contrast results from a slow drop in the laser

output energy over time, which induced a decrease in the

production of the 58Niþ. Although this behavior may appear

to be a drawback, the contrast is still sufficient to clearly

highlight the contours of the nickel microscopic grid.

Moreover, the IMS experiments were repeated several times

on different nickel grids, and the results showed that the

inter-day image quality was reproducible.


RESULTS AND DISCUSSION

The main objective of this work is to efficiently localise,

identify, and differentiate metabolites produced by Plasmo-

para viticola infection at the surface of grapevines leaves.

Three steps were undertaken to succeed in this objective.

First, the experimental analytical conditions were optimised

by considering standards of resveratrol and pterostilbene. In

the second step these compounds were detected on the

surface of a grapevine leaf. Finally, different metabolites

were localised by LDI-IMS on the surface of a grapevine leaf.

Analysis of standard resveratrol andpterostilbene compounds by LDI-MSAfter deposition at the surface of the sample holder both

resveratrol and pterostilbene were analysed by LDI-MS.

These compounds were chosen because of their importance

for grapevine leaves when plants react to biotic and abiotic

stresses. LDI-MS at 266 nm wavelength was preferred to

MALDI analysis to avoid interferences between resveratrol

and pterostilbene and ions produced by laser irradiation at

377 nm wavelength of commonly used matrices such as 2,5-

dihydroxybenzoic acid (DHB) or 4-hydroxycyanocinnamic

acid (HCCA). In addition, both these compounds are also

known to be metabolites or constituents of plants. Finally, a

restricted number of pretreatment steps is also important in

IMS to ensure quick analysis and to avoid sample alteration –

i.e. delocalisation of compounds of interest.

Both negative and positive mass spectra have been

acquired. The positive ion mode appeared to be more

suitable and allowed the detection of ions associated with

both resveratrol and pterostilbene. The positive LDI-ToF

mass spectra of both stilbenes, reported in Fig. 3, yielded

molecular radical ions at m/z 228.078 and 256.108 for

resveratrol and pterostilbene, respectively. The signal-to-

noise (S/N) ratio appeared to be higher for resveratrol. No

ions corresponding to proton, sodium or potassium adducts

of the stilbenes molecules were detected under the

experimental conditions used. The 266 nm LDI mass

spectrum of pterostilbene also yielded an intense signal at

m/z 254.094, which was assigned to the [M–2H]þ.

ion. LDI-

MS of pterostilbene was also conducted at the 337 nm

wavelength, where the absorption of pterostilbene in

methanol is greater than is generally reported at 266 nm.


DOI: 10.1002/rcm

Figure 3. LDI-ToF mass spectra of (a) resveratrol and (b)

pterostilbene; in insert, expansion around molecular cation

peaks.

338 G. Hamm et al.

The S/N ratio observed for the m/z 256.108 ion decreased by a

factor of 40 at 337 nm compared with what was obtained at

266 nm. This may be explained by considering both the

theoretical calculations and the REMPI experiments per-

formed on resveratrol. Unfortunately, no data were found for

pterosilbene. The vertical ionisation energy of resveratrol

was found to be close to 6.76 eV.31 Consequently, vertical

ionisation may be a one-color two-photon process at both 266

and 337 nm wavelengths for which the energy per photon is

4.66 and 3.68 eV, respectively. Nevertheless, REMPI exper-

iments32 have demonstrated that photoionisation in the gas

phase is optimum at the 302.1 nm wavelength, which seems

Figure 4. Photoisomerisation and phot


to indicate that the S1 excited level is 4.10 eV above the S0

ground state. Consequently, ionisation of resveratrol and to

some respect of pterostilbene is definitely more efficient at

266 nm than at 337 nm because of the inefficient S0!S1

transition at the latter wavelength.33 Consequently, 266 nm

appeared to be the more suitable desorption wavelength for

the investigation of both metabolite stilbene compounds by

mass spectrometry. Moreover, this wavelength also increases

the sensitivity for the detection of other leaf cuticle-related

compounds.34 Another interesting difference in the exper-

iments performed on pterostilbene at 266 and 337 nm is the

behavior of the [M–2H]þ.

ion detected at m/z 254.094, which

was found to be predominant at 266 nm and absent at the

337 nm wavelength. The formation of this fragment ion can

be explained by a two-step photochemical process, which

involves the conversion of a methoxylated stilbene com-

pound into a phenanthrene-like species as was observed for

tetra-methoxylated stilbene.35 In a first step, 266 nm UV

irradiation leads to a trans-cis isomerisation of pterostilbene.

Elimination of two hydrogen atoms and cyclisation of the

resulting cis-stilbene yields a polysubstituted phenanthrene

compound, which is detected at m/z 254.094 in 266 nm LDI

experiments (see Fig. 4). This process appears to be highly

wavelength- and compound-dependent, and this behavior

was never observed with resveratrol or with pterostilbene at

337 nm. A difference in the activation energy required to

induce trans-cis isomerisation as well as hydrogen atom

elimination may rationalise the experimental evidence. This

aspect is still under study.

In situ analysis of resveratrol and pterostilbenemetabolites on grapevine leavesAfter the identification of the ions detected by LDI-MS of

standard resveratrol and pterostilbene under the exper-

imental conditions used, analysis of the leaves was under-

taken. It has been reported that stilbene biosynthesis is

mainly located on the abaxial side of the leaf.13 Con-

sequently, in situ LDI-ToF experiments were conducted on

this area of the leaf. It should be noted that high laser power

densities are required in these experiments to yield a

representative mass spectrum of the species present at the

surface of the leaf. When experiments were conducted below

a given laser energy, no species were ejected from the surface

of the leaf and no ions were detected.

Figure 5 shows three mass spectra obtained with grape-

vine leaves which have undergone different treatments.

Figure 5(a) is the typical mass spectrum of a standard (no

treatment) grapevine leaf. Figures 5(b) and 5(c) report the

ocyclisation of trans-pterostilbene.


DOI: 10.1002/rcm

Figure 5. LDI-ToF mass spectra in positive ion mode of Cabernet Sauvignon leaves (a) without treatment, (b) Plasmopara

viticola-infected, and (c) irradiated. The insert shows the distribution of ions related to resveratrol (left) and pterostilbene (right).


mass spectra of a leaf after biotic (Plasmopara viticola) or

abiotic (UV-C irradiation) stress, respectively. No signals

associated with either resveratrol or pterostilbene phytoa-

lexins were observed in the no-treatment standard (Fig. 5(a)).


By contrast, significant amounts of these compounds are

highlighted in Figs. 5(b) and 5(c) by the detection of the

m/z 228.078 and 254.094 ions associated (vide supra) with

resveratrol and pterostilbene, respectively. The obtained


DOI: 10.1002/rcm

340 G. Hamm et al.

results confirm previously published studies, which report

the formation of these metabolites after abiotic36 or biotic37

stress. Depending on the nature of the stress the relative

distribution of both of these compounds is different.

Plasmopara viticola treatment appeared to yield less resver-

atrol metabolite than UV-C illumination, according to the

relative abundances of the m/z 228.078 and 254.094 ions in the

mass spectra reported in Figs. 5(b) and 5(c). Comparison of

mass spectra obtained in the study of leaf with or without

UV-C irradiation clearly indicated that resveratrol and

pterostilbene phytoalexin are not produced and accumulated

in vineyards under the effect of sunlight irradiation. The S/N

ratio of the m/z 228.078 and 254.094 ions should be

interpreted with great caution as indicative of the amounts

of studied phytoalexins. The sample-to-sample S/N vari-

ation may also be invoked to explain the observed

differences, when the mass spectra in Figs. 5(b) and 5(c)

are compared. Nevertheless, additional studies have demon-

strated that stilbene synthesis is more important after UV-C

illumination than during interaction with a pathogen.37 In

some respects, the relative ion signal (S/N ratio) may

consequently be used to compare two leaves, which have

undergone different stress or treatment before the LDI-MS

analysis. More precisely, the MS image reflects the variation

in concentration of a specific molecule in a leaf. Although

absolute quantification is not possible, the variation of

relative ion signal is reproducible and thus it allows the

comparison between two leaves. This approach also

rationalises the use of LDI instead of MALDI in which an

ion suppression effect was often reported in the mass range

close to the m/z values of the MALDI matrix ions. It should

also be noted that detection of both resveratrol and

pterostilbene on leaves submitted to biotic and abiotic stress

is not indicative of their direct or indirect implication in plant

resistance. However, a correlation between stilbene levels

Figure 6. LDI-ToF mass spectrum obtained directly from i

400. The insert shows an enlarged view of them/z 530–640

as internal calibrants.


and pathogen resistance has been established by previous

works.36

In situ analysis of leaves by LDI-MS also led to the detection

of ions at higher m/z values. More specifically, ions in the m/z

400–700 and 900–1100 ranges were clearly present. As an

example, Fig. 6 reports the mass spectrum obtained when an

untreated grapevine leaf was analysed by 266 nm LDI-MS. To

increase the level of accuracy of the mass measurements,

internal calibration was carried out with the potassium adduct

ions of PEG 600 (starred ions in the mass spectrum reported in

Fig. 6). The relative measured error was in the 1–9 ppm range.

Under these conditions, the m/z 400 to 700 ions were found to

be well described by the formulae CnH2n-4Oþ:4 (see Table 1)

which are consistent with acylium ions. The formulae are

reinforced by FTICRMS data. The observation of these

acylium ions suggests the presence of glycerides.

IMS of grapevine leavesLDI-MS allowed ions related to resveratrol and pterostilbene

to be identified. These results have been used to investigate

by LDI-IMS the spatial distribution of these secondary

metabolites in or at the surface of different kinds of grapevine

leaves. All the IMS experiments were performed using the

optimised experimental conditions defined in the previous

section. Figure 7 reports the optical and the m/z 228.078 and

254.094 LDI-MS images of control, inoculated and UV-C

irradiated grapevine leaves. Depending on the investigated

sample the size of the image varies from 0.9� 1.8 mm

(36� 72 pixels) for the infected and control leaf to 0.5� 1 mm

(19� 39 pixels) for the irradiated leaf.

The m/z 228.078 and 254.094 image of the control leaf

confirmed the results obtained by in situ LDI-MS analysis

(see Fig. 7). Indeed, no signals are observed at m/z values

associated with either the resveratrol or the dehydrogenated

pterosilbene ions. Before considering the results obtained by

noculated Cabernet Sauvignon leaf coated with PEG

region. Peaks labeled with stars are polymer ions used


DOI: 10.1002/rcm

Table 1. Main ions observed during LDI-ToFMS and LDI-FTICRMS analysis in positive ionmode of grapevine leaves and possible

elemental formula assignments. (x) denotes not detected

m/z valueAccurate mass

value (exp.)Exact mass

value (theor.)Difference

TOFMS (ppm)Difference

FTICRMS (ppm)Elementalformula

228 228.078 228.0781 0.4 � C14H12Oþ:3

254 254.094 254.09375 0.2 � C16H14Oþ:3

550 550.494 550.49556 2.3 0.5 C35H66Oþ:4

564 564.516 564.51121 8.8 �0.5 C36H68Oþ:4

578 578.527 578.52686 0.2 �1.0 C37H70Oþ:4

592 592.540 592.54251 4.7 �0.6 C38H72Oþ:4

606 606.558 606.55816 0.8 �1.5 C39H74Oþ:4

620 620.579 620.57381 8.0 3.3 C40H76Oþ:4

634 634.591 634.58946 2.7 0.1 C41H78Oþ:4

973 972.928 972.92941 1.5 �0.5 C63H120Oþ:6

1001 1000.939 1000.93924 0.1 �2.1 C65H124Oþ:6

1029 1028.971 1028.97058 0.1 �1.9 C67H128Oþ:6

Figure 7. (a) Optical images of Cabernet Sauvignon leaves irradiated, inoculated and

without treatment. MS images of two stilbenes (b) resveratrol (m/z 228.078) and (c)

pterostilbene (m/z 254.094). (For both control and inoculated leaves, image contrast scale

is the same and normalised to the highest intensity of the ion concerned. Irradiated sample

has its own contrast scale because of the very high signal intensity compared with those of

the two other samples.) Circle represents pathogen attack area.


IMS of stressed grapevine leaves, some mention has to be

made of the Plasmopara viticola pathogen infection mode.

Downy mildew is propagated under leaf tissue and passes

through stomata apertures on the abaxial side of leaf to

exhibit aerial mycelium. It is composed of sporangiophores

and sporangia which are mainly responsible for pathogen

propagation. The amounts of phytoalexins are directly


related to the degree of pathogen contamination. Optical

microscopy images obtained on a grapevine leaf inoculated

with Plasmopara viticola (Fig. 7(a), center) allowed us to

localise (circle) the aerial mycelium of the pathogen after

downy mildew attack. At this same place the m/z 228.078 and

254.094 ion images (Figs. 7(b) and 7(c), center) contrast

revealed significant amounts of resveratrol and, to a lesser


DOI: 10.1002/rcm

342 G. Hamm et al.

extent, pterostilbene. Their spatial distribution may also give

some information on the intra-propagation of pathogen in

tissue. Indeed, the distribution of the ions at m/z 228.078 and

254.094 allows the development of the mycelium near the

sporulation area to be followed.

The m/z 228.078 and 254.094 images of the UV-C-irradiated

leaf highlighted that large amounts of both investigated

stilbenes were detected on the whole surface (Figs. 7(b)

and 7(c), right). In spite of a homogeneous UV-C irradiation,

resveratrol and pterostilbene were not found to be homo-

geneously localised. Difference in their contrasts appeared

and the pterostilbene species was mainly found near or on

the veins on the abaxial surface of the leaf.

As already observed by in situ analysis, some differences in

the response of stilbene-targeted compounds at the surface of

the leaf after abiotic and biotic stress have been found.

Depending on the location, the ion signal is more intense (up

to 5500 arbitrary units) for UV-C-irradiated leaves than for

infected leaves (800 arbitrary units). However, Schmidlin

et al. reported that amounts of pterostilbene produced by

UV-C irradiation were lower than after Plasmopara viticola

infection.37 Their results are consistent with some of the IMS

results (Fig. 7(c)). Pterostilbene, which is mainly concen-

trated in the cell walls or veins of leaves after irradiation, is

thought to be more easily extracted by solvent prior to HPLC

analysis.

CONCLUSIONS

In the field of grapevine studies, the original proposed

imaging approach to directly analyse grapevine leaves by

LDI-ToFMS allows information on the nature and localis-

ation of metabolites induced by biotic (Plasmopara viticola) or

abiotic (UV-C light) stresses to be collected. Simple sample

preparation and ability to easily generate several images

with good spatial resolution (25mm) on a small leaf surface

(0.9� 1.8 mm) are the main advantages of this approach. It

was demonstrated that both resveratrol and pterostilbene

metabolites (two main compounds generated by leaves

against a stress) can be detected in situ and characterised. IMS

allows the localisation of these two compounds to be

accurately defined. After the attack by downy mildew,

LDI-ToFMS images of inoculated Cabernet Sauvignon leaf

revealed a co-localisation of resveratrol and pterostilbene

near the downy mildew lesion. When the leaf was

illuminated by UV-C light, pterostilbene was found to be

localised near or on the veins on the abaxial surface of the

leaf. Resveratrol seemed to be less well localised. Thus,

the proposed analytical protocol allows easy recognition of

the interaction between grapevine leaves and biotic or abiotic

stress to be obtained. Further works will be dedicated to

trying to identify a specific marker of downy mildew

pathogen. This work will simultaneously follow the

propagation of the pathogen and the production of defense

molecules at different stages of the infection, more particu-

larly when Plasmopara viticola has not still sporulated and is

not visible on the surface of the leaf.


AcknowledgementsThis work received financial support from the Conseil Inter-

professionnel du Vin de Bordeaux (CIVB, Bordeaux, France).

The authors thank Dr Frederic Aubriet and Pr. Patrick

Chaimbault for fruitful discussions. We are grateful to the

team of INRA Colmar laboratory for assistance with plant

material, Dr Lionel Vernex-Loset for technical assistance and

Stefen Clerens for his help on software support.

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DOI: 10.1002/rcm

Determination and imaging of metabolites from Vitis vinifera leaves by laser desorption/ionisation...

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