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Ethyl gallate displays elicitor activities in tobacco plants Pascale GOUPIL, Razik Benouaret, and Claire Richard J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03051 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on October 2, 2017

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Journal of Agricultural and Food Chemistry

Title: Ethyl gallate displays elicitor activities in tobacco plants

Authors: Pascale Goupil1,2 *, Razik Benouaret1,2, Claire Richard3,4

Affiliations: 1

Université Clermont Auvergne, UMR 547-UBP/INRA PIAF, Campus universitaire des Cézeaux, 8 avenue Blaise Pascal, 63178 Aubière cedex, France, 2

INRA, UMR 547 PIAF, Clermont-Ferrand, France

3

Université Clermont Auvergne, CNRS, UMR 6296, Campus universitaire des Cézeaux, Institut de Chimie de Clermont-Ferrand (ICCF), 24 avenue Blaise Pascal, 63178 Aubière cedex, France

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Equipe Photochimie CNRS, UMR 6296, ICCF, 63178 Aubière, France

*Author for correspondence: Pascale Goupil Phone: (33) 04 73 40 79 40 Fax: (33) 04 73 40 79 42 E-mail: [email protected]

Running title: Alkyl gallates in plant defence

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ACS Paragon Plus Environment


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Abstract

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Alkyl gallates showed elicitor activities on tobacco in both whole plants and cell suspensions.

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Methyl gallate (MG), ethyl gallate (EG) and propyl gallate (PG) infiltration into tobacco

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leaves induced hypersensitive reaction-like lesions and topical production of autofluorescent

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compounds revealed under UV light. When sprayed on tobacco plants at 5 mM, EG promoted

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upregulation of defence-related genes such as the antimicrobial PR1, -1,3-glucanase PR2,

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chitinase PR3 and osmotin PR5 target genes. Tobacco BY-2 cells challenged with EG

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underwent cell death in 48 h, significantly reduced in the presence of the protease inhibitor

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aprotinin. The three alkyl gallates all caused alkalinisation of the BY-2 extracellular medium,

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whereas gallic acid did not trigger any pH variation. Using EGTA or LaCl3, we showed that

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Ca2+ mobilisation occurred in BY-2 cells elicited with EG. Overall, our findings are the first

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evidence of alkyl gallate elicitor properties with early perception events on plasma membrane,

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potential hypersensitive reactions and PR-related downstream defence responses in tobacco.

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Key words: Alkyl gallates, BY-2, Elicitors, Ethyl gallate, Pathogenesis-related protein, Plant

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defence reactions, Tobacco

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Introduction

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Open-field crops are always vulnerable to pathogens, and so extensive farming requires

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constant control of pathogen populations. Synthetic pesticides are commonly used for direct

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pest control, but need repeated spreading for season-long protection. Pesticide action is

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mostly selective and requires combinations of active compounds in an integrated pest

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management approach. A promising alternative that emerged in the 1990s is based primarily

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on improving the endogenous defence capabilities of plants. When plants are attacked by

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pathogens, they defend themselves with an arsenal of defence mechanisms. These active plant

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defence responses comprise three successive events: (i) recognition of the pathogen, (ii) signal

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transduction, and (iii) execution of the defence programmes with activation of defence genes.1

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The recognition events involve pathogen- or plant-derived signalling entities termed elicitors.2

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Intracellular signal conversion and transduction include the very early elicitor-induced

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signalling processes such as protein phosphorylation, influx of Ca2+, efflux of H+, K+ and Cl−

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leading to extracellular alkalinisation, depolarisation of the plasma membrane, and active

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oxygen species production, culminating in the hypersensitive response, a localised cell

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suicide.3 Ultimately, the pathogen challenge leads to the production of phytoalexins and

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pathogenesis-related proteins, cell wall and/or cuticle reinforcement which prevent pathogen

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development. Point inoculation of the pathogen, in addition to triggering the local acquired

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resistance response (LAR), can mobilise systemic acquired resistance (SAR), which alerts and

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predisposes the tissues to a more intensive response on subsequent infection.1,4 Salicylic acid

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(SA) plays a crucial role in the SAR induction processes. SA is involved in the signal

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transduction systems by activating enzyme expression catalysing biosynthetic reactions to

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produce defence compounds 5,6 and provide protection for plants against pathogens.

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As it is pathogenesis that initiates SAR, impact on disease is lower than if the SAR response

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had been activated before the arrival of the pathogen. The use of plant defence inducers 3



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(PDIs) to establish SAR intentionally and preventively in plants are thus currently a promising

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strategy in phytoprotection. PDIs are organic, inorganic, botanical or synthetic stimulus-

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triggering factors (elicitors) that mobilise plant defence reactions.7,8 By stimulating plant

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immunity, PDIs provide defence against subsequent infection.7,9 To date, only very few

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substances with high SAR activity have been described 9, and they mostly combine PDI and

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pesticide actions, not necessarily a disadvantage since both properties are inimical to

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pathogens. 10

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Agricultural applications of plant activators are far from valued.9 The synthetic agrochemicals

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now available are SA-like substances that mimic the action of this phytohormone. Of these,

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2,6-dichloro isonicotinic acid (INA) and acibenzolar-S-methyl (BTH) and their derivatives are

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the most thoroughly studied, currently marketed as BOOST®, Bion® and Actigard™. These

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resistance activators serve as efficient chemical inducers of SAR, with effectiveness against

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diverse plant diseases.11,12

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The identification of new chemicals able to induce disease resistance is beneficial not only for

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elucidating the pathways leading to SAR, but also for developing new plant protection agents.

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The effectiveness of various elicitors depends on plant taxonomy, and there are many

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examples where an elicitor is more effective on some plant species than on others.8 We have

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been engaged in seeking naturally-occurring or synthetic substances able to induce SAR

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reactions. As part of this research we evaluated the PDI activity of alkyl gallates (3,4,5-

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trihydroxybenzoates) on tobacco plants. Gallic acid derivatives had already been found to

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display antimicrobial properties. Octyl, nonyl and lauryl gallates exhibit fungicidal and

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bacterial activity.13,14 Silva et al.15 showed their potential for use in disease management,

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evaluating the inhibitory activity of a set of gallic acid alkyl esters on the plant pathogenic

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bacterium Xanthomonas citri. Here we demonstrate for the first time that alkyl gallates act at

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various points in the signalling pathway leading to defence reactions. We report evidence that 4


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treatment of tobacco with methyl, ethyl and n-propyl gallates induces hypersensitive-like

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reactions with activation of defence-related genes in the tobacco plant. These gallic acid

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derivatives trigger rapid extracellular alkalinisation of BY-2 plant cells with Ca2+

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mobilisation, suggesting that tobacco cells perceive the chemicals by modifying plasma

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membrane physical properties. Finally, the naturally-occurring and semisynthetic gallic acid

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derivatives revealed potent PDI actions, revealing a valuable source of compounds that could

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find use in green agriculture practices.

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Materials and methods

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Biological compounds and chemicals

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Methyl gallate (MG), ethyl gallate (EG), n-propyl gallate (PG), salicylic acid (SA), and gallic

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acid (GA) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). All the

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compounds were dissolved in ultrapure water (MilliQ). PG needed warm (25 °C) ultrapure

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water to dissolve completely.

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Plant material and treatments

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The biological activity of alkyl gallates was measured on 3-month old tobacco plants

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(Nicotiana tabacum L.) bearing 20–22 leaves (preflowering stage). Tobacco plants were

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grown in a greenhouse under controlled conditions (22 ± 5 °C with a photoperiod of 16 h

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light). The chemical solutions to be tested (50 µl) were infiltrated into the leaf tissue using a

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plastic syringe (without a needle), on an area of 1–2 cm2. As a positive control, leaves were

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infiltrated with 2 mM SA. For the negative control, leaves were infiltrated with ultrapure

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water. The compounds were infiltrated into distinct areas on the same leaf for the examination 5



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of macroscopic symptoms under bright field or UV light (at 312 nm). For PR transcript

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quantification, three mature leaves were sprayed on both their upper (adaxial) and lower

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(abaxial) surfaces. Foliar spray treatments were administered with a fine atomiser until the

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solutions dripped (3 mL per leaf on average). The negative control was leaves sprayed with

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ultrapure water.

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Tobacco cell suspensions of Nicotiana tabacum L. cv. Bright Yellow-2 (BY-2) were grown in

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MS medium (Duchefa, Netherlands) supplemented with sucrose (30 g.L-1), thiamine (1 mg.L-

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1

), myo-inositol (102 mg.L-1) and 2,4-dichlorophenoxyacetic acid (0.2 mg.L-1) at pH 5.8. Cells

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were agitated in the dark (140 rpm, 25 °C) and subcultured weekly by dilution at a 1:15 ratio

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in fresh medium. For determination of extracellular pH changes, cells from culture in

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exponential-phase growth were collected by filtration, washed with 175 mM mannitol, 0.5

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mM CaCl2, 0.25 mM MgCl2, 1 mM KCl and 1 mM sucrose, and resuspended at 0.1 g fresh

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weight (FW)/mL in the same medium. The initial pH was about 4.8.

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Semi-quantitative RT-PCR

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The patterns of PR gene expression were analysed by real-time quantitative PCR (RT-qPCR)

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using SYBR-green and the iCycler iQv5 (Bio-Rad). Total RNA was extracted from tobacco

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leaf tissue (200 mg) in cetyltrimethylammonium bromide extraction buffer, as described by

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Chang et al.16. Total RNA was cleaned up with 0.5 U DNase I solution (Euromedex, France)

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containing 20 U RNase inhibitor (Euromedex, France). RNA integrity was verified on a 1%

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agarose gel by detecting ribosomal RNA. RNA concentrations were measured

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spectrophotometrically at 260 nm. First-strand cDNA was synthesised from up to 1 µg of total

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RNA with Euroscript Reverse Transcriptase (Eurogentec, France) according to the

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manufacturer’s instructions. PCR reactions were prepared using the qPCR kit Mastermix for 6


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SYBR green (Eurogentec) according to the manufacturer’s protocol. The cDNA concentration

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used produced a threshold cycle value (CT) of between 15 and 30 cycles. Amplification

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specificity was checked by melting-curve analysis. The relative quantity (QR) of PR gene

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transcripts using the EF-1α gene

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mathematical model.18 Values are given as log2QR. For every PR gene analysed, three

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independent biological replicates were run, and every run was carried out at least in triplicate.

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Primers and amplicon sizes are given in Benouaret and Goupil.19

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as internal standard was calculated with the delta-delta

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BY-2 Extracellular alkalinisation

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The pH variations of the culture medium were recorded by introducing a glass microelectrode

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(Hanna Instrument, HI1330B, France) into 6 ml of the equilibrated cell suspension culture.

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The pH was measured every 10 min starting immediately after treatment with elicitors or

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ultrapure water (negative control). The ∆pH was the pH measured every 10 min relative to the

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pH measured at t0 immediately after adding elicitors or ultrapure water. Each experiment was

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repeated at least three times to check reproducibility. Figures depict the results of typical

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experiments. Pharmacological treatments were conducted using the chelator EGTA (2 mM)

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and the Ca2+ channel blocker LaCl3 (2 mM).

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Cell death measurement

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The Evans Blue assay was performed according to Amano et al. (2003) using a plastic syringe

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device. The pigment enters cells with damaged plasmalemma. The assay was first calibrated

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with a set of viability standards using mixtures of living and dead tobacco BY-2 cells

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(Benouaret et al., 2014). Tobacco cells (500 µL) were withdrawn from the culture medium

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immediately (t0) and then 24 h after elicitation. Pharmacological treatment was conducted

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with the serine protease inhibitor aprotinin (0.2 µM) 30 min before cell death rate

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determination. Absorbance at 600 nm was measured to estimate cell death. Each experiment

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was repeated three times with three independent BY-2 cultures.

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Results

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Macroscopic changes of tobacco tissues challenged with alkyl gallates

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Foliar tobacco tissues were infiltrated with the three alkyl gallates (Fig. 1) at 5 mM, and the

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macroscopic changes were examined under bright and UV light. Fig. 2 shows the kinetics of

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appearance of macroscopic symptoms on the abaxial face of tobacco leaves exposed to bright

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light (Fig. 2A-D) and UV light (Fig. 2E-H). UV light examination (λ = 312 nm) revealed that

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the methyl gallate (MG)-, ethyl gallate (EG)- and n-propyl gallate (PG)-infiltrated zones and

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the surrounding tissues displayed autofluorescence indicative of the accumulation of phenolic

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compounds (Fig. 2F-H). At 2 and 4 dpi, tobacco mesophyll infiltrated with MG was

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unchanged under bright light, but the chemical induced autofluorescence in the surrounding

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tissues. A restricted chlorotic zone appeared at 8 dpi without production of fluorescent

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compounds. By contrast, the EG-infiltrated tissues turned chlorotic by 4 dpi and tissue injury

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was enlarged at 8 dpi. The surrounding zone was loaded with phenolic compounds. The PG-

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infiltrated tissues showed the most pronounced signs, with restricted brown areas at 2 dpi and

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a brown extended zone at 4 and 8 dpi. Examination of the leaf tissue by UV light revealed that

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the necrotic zone was surrounded by a scattered pattern of fluorescence. Overall, tobacco

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leaves were receptive to the three alkyl gallates tested. Since the alkyl gallates themselves did

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not emit fluorescence (Fig. 2E), these results evidence that the chemical compounds induced

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marked metabolic changes over the interveinal areas of the leaves. The naturally-occurring 8


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EG was retained for further work, since it proved highly active in inducing accumulation of

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autofluorescent compounds.

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A dose-response experiment was undertaken on EG-infiltrated tobacco tissues. Macroscopic

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symptoms were examined under bright and UV light for EG concentrations ranging from 5

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mM to 0.5 mM. Fig. 3 shows the infiltrated areas at 4 dpi. The 5 mM-infiltrated zone

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appeared slightly chlorotic and displayed a large fluorescent surrounding area. Expansion of

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autofluorescence was induced dose-dependently. EG infiltration at 2 mM resulted in faint

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autofluorescence, and at 1 mM had no effect. As expected, SA (2 mM) induced a necrotic

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area with local accumulation of fluorescent compounds.

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Responses of cultured cells to alkyl gallates

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We used medium alkalinisation as a readily measurable effect on suspension-cultured BY-2

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cells to test the potential elicitor activity of alkyl gallates. As shown in Fig. 4A, alkalinisation

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of the culture medium in response to AG was evidenced for the 1 mM and 5 mM

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concentrations. Suspended cells responded to AG by a rapid, sustained alkalinisation of the

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incubation medium. Extracellular alkalinisation was detected immediately after adding the

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AG. The three alkyl gallates induced a ∆pH reaching 0.8 to 1.1 units at 1 mM and 1.5 to 1.8

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units at 5 mM after 150 min. Fig. 4B shows that the extent of pH variation was dose-

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dependent in the concentration range used for EG. The threshold value was not reached in our

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experimental system; the lowest concentration (0.5 mM) could still cause a pH change of 0.8

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units. As expected, no pH shift was detected with the control cells treated with water.

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The calcium mobilisation in EG-induced pH changes was investigated by adding the chelator

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agent EGTA (2 mM) and the Ca2+-channel blocker LaCl3 (2 mM) to the culture medium

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concomitantly with the EG. Both chemicals radically suppressed the pH shift. Adding the

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chemicals during the alkalinisation phase, i.e. 30 min after elicitation, resulted in rapid

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abolition of the pH rise. EDTA and LaCl3 had an inverse effect on the extracellular pH

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medium in control cells, with slight medium acidification (Fig. 4C).

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The eliciting activity of gallic acid (GA) was measured by medium pH determination.

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Addition of GA at 0.5 mM and 5 mM to the BY-2 cell suspension did not trigger any pH

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variation in the extracellular medium, suggesting that the compound was not perceived by the

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tobacco cells, at least as noted above (Fig. 4D).

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The EG-triggered hypersensitive-like response was investigated in BY-2 cells using the Evans

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blue test (Fig. 5). Cell death rate was measured in tobacco cells exposed to EG at 0.5 mM,

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2 mM and 5 mM for 48 h (Fig. 5A). Cell death rate was 10% on average in control cells

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(without EG). The EG elicitation induced significantly higher cell death. The exposure of

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tobacco cells to 2 mM and 5 mM EG increased the magnitude of the cell death rate, which

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reached 25% and 45% respectively. The action of the plant cell death inhibitor aprotinin was

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examined on 5 mM EG-elicited cells. As shown in Fig. 5B, the protease inhibitor lowered cell

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death rate by 19%, whereas the inhibitor did not affect cell viability in control tobacco cells.

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Ethyl gallate-induced PR transcript accumulation

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The ability of EG to induce defence-related gene expression was investigated in tobacco

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plants. Transcript levels of four PR target genes were quantified by quantitative real-time

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PCR in EG-treated leaves 4 days after spraying. At 1 mM, EG failed to induce significant

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transcript accumulation of the four PR markers. At 5 mM, EG led to high PR transcript

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accumulation: 60-fold for the antimicrobial PR1, 50-fold for β-1,3-glucanase PR2, 35-fold for

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chitinase PR3, and 30-fold for osmotin PR5, on average (Fig. 6).

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Discusion

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The present work demonstrates the ability of three alkyl gallates to elicit plant defence

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reactions. Using tobacco as a plant model, we show the potential of methyl gallate (MG),

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ethyl gallate (EG) and n-propyl gallate (PG) to trigger local injuries, biochemical changes and

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PR transcript accumulation. Infiltration of tobacco leaves with the chemicals resulted in foliar

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symptoms and local accumulation of fluorescent compounds. The three compounds induced

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restricted chlorotic tissues visualised under bright light, with production of phenolics in the

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surrounding areas, suggesting cell death initiation of the mesophyll cells. The topical

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reactions induced by the three alkyl gallates were of different severity in terms of tissue injury

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and production of phytoalexin-like entities. The fact that the intensity of effects varied with

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the alkyl gallates (MG < EG < PG) suggests that the compounds were active to different

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degrees at the 5 mM concentration, or that their modes of action differed in the necrotic

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reaction associated with cell death. EG and PG seemed to be the most potent inducers of local

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cell death in our experiment. Localised cell death in plant tissue resulting from attack by

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pathogens can occur either in hypersensitive resistant response or in harmful reactions such as

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necrosis potentially caused by toxins.22 We demonstrate here that BY-2 cell death is sensitive

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to protease inhibitor, suggesting that infiltrated tobacco tissues may evolve towards

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hypersensitive status. However, phytotoxic reactions caused by alkyl gallate cannot be ruled

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out.

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Rapid changes in membrane permeability are involved in plant defence-associated early

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responses to various biotic elicitors.23 Increased influx of protons and calcium mobilisation

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are both early events in the elicitor signalling network. Perception of alkyl gallates visualised

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by the pH rise in the incubation medium was evidenced on BY-2 cells. Medium alkalinisation

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is one of the very early events occurring in elicitor-binding sites in plant cells 24, and results

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from elicitor-induced depolarisation of the plasma membrane with subsequent K+/H+

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exchanges and Ca2+ influx/Cl− efflux. In our experiments, the effect of Ca2+ influx inhibitors

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EGTA or LaCl3 caused the decreased pH shift of the extracellular medium, suggesting that

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Ca2+ mobilisation occurred in BY-2 cells elicited with alkyl gallates. As a consequence of ion

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fluxes, a predictive cytoplasmic acidification should take place rapidly in BY-2 cells.

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Cytoplasmic acidification is regarded as an essential step in signal transduction, leading to

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early defence gene expression and secondary metabolite biosynthesis.25

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Kinetics of extracellular pH changes vary among elicitors, with two main different behaviours

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towards cells. According to the nature of the elicitor, the perception can induce a long-

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sustained pH rise, or the pH variation can be transient, with a return to the resting value. It is

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noteworthy that the modification of H+ flux in BY-2 cells following treatment by alkyl

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gallates was similar to the defence response elicited by a polyphenolic-enriched grape

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extract.21 The induced H+ flux modification lasting 150 min presented similar characteristics

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in the respective time course of the effect. This result suggests that the underlying processes

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are basically the same. EG is a naturally-occurring gallic acid derivative found in several plant

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sources and identified in wines from different varieties of Vitis vinifera.26 It was not detected

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in a red winery by-product extract (data not shown), suggesting that this biomolecule is not

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active in grape marc extract.

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Alkyl gallates are antioxidants. One of the earliest cellular responses following elicitor

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recognition is oxidative burst with production of active oxygen species (AOS). Despite their 12


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damaging effect, AOS are well-described second messengers producing plant responses.

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Whether AOS will act as a destructor or as a signalling entity depends on the equilibrium

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between AOS production and scavenging. Low concentrations of AOS act as signalling

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entities mediating the plant responses: however, high concentrations cause acute damage to

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cell components.27 In elicitor-challenged cells, disruption in cellular redox homeostasis appear

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to switch on a signalling cascade leading to programmed cell death, encountered during

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hypersensitive response (HR).28 Using pharmacological approaches, our group came near to

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the hypersensitive status of BY-2 cells that led to cell death on EG treatment. In this BY-2

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system, the antioxidant gallates acting as free radical scavengers could subsequently help to

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reduce physiological AOS equilibrium and induce HR-related cell death. The potential ability

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of phenolics to induce HR-related cell death has been previously demonstrated with

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polyphenolic-enriched grape marc extracts.21 Even though EG was not detected in the grape

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marc extracts, it can be assumed that the antioxidant properties of polyphenolics could be

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involved in HR establishment. Investigation of AOS as potential agents with pharmacological

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approaches will help to characterise the pathways leading to the hypersensitive status of EG-

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induced cell death in tobacco.

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Alkyl gallates evidently activate the SAR pathway, since they induced expression of the same

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set of SAR genes, i.e. PR1, PR2, PR3 and PR5, that are induced by SA.29 Numerous synthetic

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and/or bio-based elicitors can induce plant immune responses. The most marked chemical

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ones, acibenzolar-S-methyl (ASM) and 2,6-dichloroisonicotinic acid (INA), both induce SAR

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without SA accumulation; probenazole (PBZ) induces SAR through SA biosynthesis.11,30 SA

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involvement in alkyl gallate-regulated plant defences and the potential of these compounds to

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protect cultivated crops are being investigated in laboratory studies.

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In conclusion, the present work offers evidence for plant immunity inducer properties of alkyl

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gallates. Plants can synthesise a wide range of phenolics as an integral part of their non-host 13



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resistance strategy. These secondary metabolites are natural defensive antibiotics, ensuring

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efficient chemical barriers to pathogens. Their elicitation properties are demonstrated here for

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the first time in alkyl gallates, which emerge as PDI agents suited to phytoprotection. Being

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naturally-occurring and ubiquitous, EG is the most promising of these agents. It is potentially

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non-toxic for the human being, and offers consumer benefits, with antioxidant, anti-

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inflammatory and anticarcinogenic activities. Given that EG can play a dual role in plant

288

protection, as it also possesses direct antimicrobial activities, this biocontrol agent constitutes

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a potent product well-suited to environmentally-oriented schemes for high crop protection.

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Acknowledgements

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This work was supported by grants from FUI: Fonds Unique Interministériel.

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The authors thank Céline Sac for help with the tobacco cell cultures and laboratory assistance,

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and Dominique Marcon for technical assistance in photographic editing.

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References

300 301 302 303 304 305

1. Fu, Z.Q.; Dong, X. Systemic acquired resistance: turning local infection into global

306

defense. Annu. Rev. Plant Biol. 2013, 64, 839-863.

307

2. Choi, H.W.; Klessig, D.F. DAMPS, MAMPS, and NAMPS in plant innate immunity. BMC

308

Plant Biol. 2016, 16, 232. doi:10.1186/s12870-016-0921-2.

309

3. Yang, Y.; Shah, J.; Klessig, D.F. Signal perception and transduction in plant defence

310

responses. Genes Dev. 2011, 11, 1621-1639.

311

4. Kachroo, A.; Robin, G.P. Systemic signaling during plant defense. Curr. Opin. Plant Biol.

312

2013, 16, 527-533.

313

5. Chen, Z.; Zheng, Z.; Huang, J.; Lai, Z.; Fan, B. Biosynthesis of salicylic acid in plants.

314

Plant Signal Behav. 2009, 4, 493–496.

315

6. Boatwright, J.L.; Pajerowska-Mukhtar, K. Salicylic acid: an old hormone up to new tricks.

316

Mol. Plant Pathol. 2013, 14, 623-634.

317

7. Walters, D.R., Ratsep, J., Havis, N.D. Controlling crop diseases using induced resistance:

318

challenges for the future. J. Exp. Bot. 2013, 64, 1263-1280.

319

8. Lyon, G. Agents that can elicit induced resistance. In: Induced resistance for plant defence.

320

A sustainable approach to crop protection. Walters, D.R.; Newton, A.; Lyon, G. eds.;

321

Blackwell Publishing Ltd, Oxford, 2014; 11-40.

322

9. Gozzo, F.; Faoro, F. Systemic acquired resistance (50 years after discovery): moving from

323

the lab to the field. J. Agric. Food Chem. 2013, 61, 12473-12491. 15



324

325

10. Dewen, Q.; Yijie, D.; Yi, Z., Shupeng, Li.; Fachao, S. Plant immunity inducer

326

development and application. Mol. Plant Microbe Interact. 2017, 30, 355-360.

327

11. Bektas, Y.; Eulgem, T. Synthetic plant defense elicitors. Front. Plant Sci. 2015, 5, 804.

328

doi: 10.3389/fpls.2014.00804.

329

12. Dufour, M-C.; Magnin, N.; Dumas, B.; Vergnes, S.; Corio-Costet, M-F. High-throughput

330

gene-expression quantification of grapevine defense responses in the field using microfluidic

331

dynamic arrays. BMC Genomics 2016, 17, 957. doi :10.1186/s12864-016-3304-z.

332

13. Kubo, I.; Xiao, P.; Fujita, K. Anti-MRSA activity of alkyl gallates. Bioorganic &

333

Medicinal Chemistry Letters 2002, 12, 113-116.

334

14. Leal, P.C.; Mascarello, A.; Derita, M.; Zuljan, F.; Nunes, R.J.; Zacchino, S.; Yunes, R.A.

335

Relation between lipophilicity of alkyl gallates and antifungal activity against yeasts and

336

filamentous fungi. Bioorg. Med. Chem. Lett. 2009, 19, 1793-1796.

337

15. Silva, I.C.; Regasini, L.O.; Petrônio, M.S.; Silva, D.H.S.; Bolzani, V.S.; Belasque, J.;

338

Sacramento, L.V.S.; Ferreira, H. Antibacterial activity of alkyl gallates against Xanthomonas

339

citri subsp. Citri. J. Bacteriol. 2013, 195, 85-94.

340

16. Chang, S.; Puryear, J.; Cairney, J. A simple and efficient method for isolating RNA from

341

pine trees. Plant Mol. Biol. Report. 1993, 11, 113-116.

342

17. Peng, J-L., Bao, Z-L., Ren, H-Y., Wang, J-S., Dong, H-S. Expression of harpinXoo in

343

transgenic tobacco induces pathogen defense in the absence of hypersensitive cell death.

344

Phytopathology 2004, 94,1048–1055.

345

18. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. 16


Page 16 of 27

Page 17 of 27


346

Nucleic Acids Res. 2001, 29, 2002-2007.

347

19. Benouaret, R.; Goupil, P. Grape marc extract-induced defence reactions and protection

348

against Phytophthora parasitica are impaired in NahG tobacco plants. J. Agric. Food Chem.

349

2015, 63, 6653-6659.

350

20. Amano, T.; Hirasawa, K-I.; O’Donohue, M.; Pernolle, J.C.; Shioi, Y. A versatile assay for

351

the accurate, time-resolved determination of cellular viability. Anal. Biochem. 2003, 314, 1-7.

352

21. Benouaret, R.; Goujon, E.; Goupil, P. Grape marc extract causes early perception events,

353

defence reactions and hypersensitive response in cultured tobacco cells. Plant Physiol.

354

Biochem. 2014, 77, 84-89.

355

22. Iakimova, E.T.; Michalczuk, L.; Woltering, E.J. Hypersensitive cell death in plants- Its

356

mechanisms and role in plant defence against pathogens. J. Fruit Ornam. Plant Res. 2005, 13,

357

135-158.

358

23. Garcia-Brugger, A.; Lamotte, O.; Vandelle, E.; Bourque, S.; Lecourieux, D.; Poinssot, B.;

359

Wendehenne, D.; Pugin, A. Early signaling events induced by elicitors of plant defenses. Mol.

360

Plant Microbe Interact. 2006, 19, 711-724.

361

24. Zhao, J.; Davis, L.C.; Verpoorte, R. Elicitor signal transduction leading to production of

362

plant secondary metabolites. Biotechnol. Adv. 2005, 23, 283-333.

363

25. Sakano, K. Metabolic regulation of pH in plant cells: role of cytoplasmic pH in defense

364

reactions and secondary metabolism. Int Rev Cytol. 2001, 206, 1-44.

365

26. Monagas, M.; Suárez, R.; Gómez-Cordovés, C.; Bartolomé, B. Simultaneous

366

determination of nonanthocyanin phenolic compounds in red wines by HPLC-DAD/ESI-MS.

367

Am. J. Enol. Vitic. 2005, 56, 139-147.

17



368

27. Sharma, P.; Jha, A.B.; Dubey, R.S.; Pessarakli, M. Reactive oxygen species, oxidative

369

damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot.

370

2012, 2012, 1-26.

371

28. Van Breusegem, F.; Dat, J.F. Reactive oxygen species in plant cell death. Plant Physiol.

372

2006, 141, 384-390.

373

29. Cordelier, S.; de Ruffray, P.; Fritig, B.; Kauffmann, S. Biological and molecular

374

comparison between localized and systemic acquired resistance induced in tobacco by a

375

Phytophthora megasperma glycoprotein elicitin. Plant Mol. Biol. 2003, 51, 109-118.

376

30. Burketova, L.; Trda, L.; Ott, P.G.; Valentova, O. Bio-based resistance inducers for

377

sustainable plant protection against pathogens. Biotechnol. Adv. 2015, 33, 994-1004.

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Figure captions

380

381

Fig. 1. Structures of the active ingredients studied

382

383

Fig. 2. Macroscopic symptoms induced in tobacco leaves by methyl gallate (MG), ethyl

384

gallate (EG) and n-propyl gallate (PG) infiltration observed under bright field (A-D) and UV

385

light (E-H) at 0 dpi (A,E), 2 dpi (B,F), 4 dpi (C,G) and 8 dpi (D,H). Alkyl gallates were

386

infiltrated at 5 mM. Bars 2 cm

387

388

Fig. 3. Macroscopic symptoms induced in tobacco leaves by EG infiltration observed under

389

bright field (A) and UV light (B) at 4 dpi. Tobacco leaves were infiltrated with different EG

390

concentrations: 5 mM (1), 4 mM (2), 3 mM (3), 2 mM (4), 1 mM (5) and 0.5 mM (6),

391

ultrapure water (control), 2 mM SA. Bars 1 cm

392

393

Fig. 4. Extracellular alkalinisation in tobacco BY-2 suspension cells over time elicited with

394

methyl gallate (MG), ethyl gallate (EG), and n-propyl gallate (PG). A. Alkyl gallates used at 5

395

and 1 mM, B. EG in the presence of Ca2+ inhibitors. EGTA and LaCl3 at 2 mM were added to

396

the BY-2 medium at the onset of elicitation or at 30 min (arrow), C. EG added at 0.5 to 5 mM,

397

D. Gallic acid (GA) and EG used at 5 and 0.5 mM. The control is water. The data are from

398

representative experiments out of a total of five.

399

19



400

Fig. 5. Cell death rate assessed by Evans Blue staining of tobacco BY-2 cell suspension

401

treated with ethyl gallate (EG) at 0.5, 2 and 5 mM for 48 h. The protease inhibitor aprotinin

402

was added 30 min before cell death measurement in BY-2 cells treated with water (control) or

403

5 mM EG. Values are expressed relative to control (water treatment) values. The experiments

404

were performed in triplicate with three different BY-2 cultures. Bars represent means ±SEM.

405

Letters indicate significant differences between treated and control samples according to one-

406

way ANOVA followed by LSD test (p ≤ 0.05). Bars represent the mean values ± SE, p ≤ 0.05.

407

408

Fig. 6. PR transcript accumulation in tobacco leaves 4 days after 2 mM SA (positive control)

409

or 1 and 5 mM ethyl gallate (EG) spraying. Transcripts were quantified by real-time RT-PCR

410

in the sprayed leaves. Values are expressed relative to control (water treatment) values. Three

411

leaves per plant were treated for each experiment. Each independent experiment was

412

performed in triplicate. Bars represent means ± SEM. Letters indicate significant differences

413

between treated and control samples according to one-way ANOVA followed by LSD test

414

(p ≤ 0.05).

415

416

417

418

419

420

421

20


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Page 21 of 27


Fig 1

Methyl gallate

Ethyl gallate


Propyl gallate


Page 22 of 27

Fig 2

MG

MG

MG

MG

EG

EG

EG

EG

PG

PG

PG

PG

A

B

D

C

MG

MG

MG

MG

EG

EG

EG

EG

PG

PG

PG

PG

E

F


G

H

Page 23 of 27


Fig 3

CONTROL

1

1

2

2

3

CONTROL

3

4 SA 2mM

A

SA 2mM

4

5

5

6

6

B



Fig 4

A

B

C

D ACS Paragon Plus Environment

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c

Fig 5 b a a

A b c

a

a

B ACS Paragon Plus Environment


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Fig 6

a b

Rela9ve transcript quan99es

70 60 50

PR1

40

b

30

0

PR2 PR3

20 10

b

c c c c Control

c

c

PR5

c c

EG 1mM

EG 5mM


Page 27 of 27


TOC

MG EG PG

PLANT DEFENSES REACTIONS in tobacco leaves and BY-2 -macroscopic symptoms -PR transcripts accumulation -cell death -perception events on membranes


Ethyl Gallate Displays Elicitor Activities in Tobacco Plants - Journal of

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