Ethyl Gallate Displays Elicitor Activities in Tobacco Plants - Journal of

Sep 29, 2017 - Alkyl gallates showed elicitor activities on tobacco in both whole plants ... plants at 5 mM, EG promoted upregulation of defense-relat...
0 downloads 0 Views 2MB Size
Subscriber access provided by UNIV OF ESSEX

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

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

4

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

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

Abstract

2 3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

16

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

17

defence reactions, Tobacco

18

19

2

ACS Paragon Plus Environment

Page 2 of 27

Page 3 of 27

Journal of Agricultural and Food Chemistry

20

Introduction

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

transduction systems by activating enzyme expression catalysing biosynthetic reactions to

42

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

43

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

44

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

45

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

46

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

47

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

48

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

49

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

50

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

51

pathogens. 10

52

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

53

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

54

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

55

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

56

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

57

diverse plant diseases.11,12

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

ACS Paragon Plus Environment

Page 4 of 27

Page 5 of 27

Journal of Agricultural and Food Chemistry

70

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

71

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

72

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

73

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

74

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

75

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

76

find use in green agriculture practices.

77

78

Materials and methods

79

Biological compounds and chemicals

80

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

81

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

82

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

83

water to dissolve completely.

84

85

Plant material and treatments

86

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

87

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

88

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

89

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

90

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

91

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

92

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

93

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

94

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

95

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

96

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

97

ultrapure water.

98

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

99

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

100

1

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

101

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

102

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

103

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

104

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

105

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

106

107

Semi-quantitative RT-PCR

108

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

109

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

110

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

111

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

112

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

113

agarose gel by detecting ribosomal RNA. RNA concentrations were measured

114

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

115

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

116

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

ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27

Journal of Agricultural and Food Chemistry

117

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

118

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

119

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

120

transcripts using the EF-1α gene

121

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

122

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

123

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

17

as internal standard was calculated with the delta-delta

124

125

BY-2 Extracellular alkalinisation

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

135

Cell death measurement

136

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

137

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

138

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

139

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

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

140

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

141

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

142

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

143

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

144

145

Results

146

Macroscopic changes of tobacco tissues challenged with alkyl gallates

147

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

148

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

149

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

150

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

151

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

152

the surrounding tissues displayed autofluorescence indicative of the accumulation of phenolic

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

ACS Paragon Plus Environment

Page 8 of 27

Page 9 of 27

Journal of Agricultural and Food Chemistry

164

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

165

autofluorescent compounds.

166

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

167

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

168

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

169

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

170

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

171

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

172

area with local accumulation of fluorescent compounds.

173

174

Responses of cultured cells to alkyl gallates

175

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

176

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

177

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

178

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

179

incubation medium. Extracellular alkalinisation was detected immediately after adding the

180

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

181

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

182

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

183

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

184

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

185

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

186

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

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

204

Ethyl gallate-induced PR transcript accumulation

205

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

206

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

207

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

208

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

10

ACS Paragon Plus Environment

Page 10 of 27

Page 11 of 27

Journal of Agricultural and Food Chemistry

209

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

210

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

211

212

213

Discusion

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

out.

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

early defence gene expression and secondary metabolite biosynthesis.25

244

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

245

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

246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

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

254

active in grape marc extract.

255

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

256

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

ACS Paragon Plus Environment

Page 12 of 27

Page 13 of 27

Journal of Agricultural and Food Chemistry

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

induced cell death in tobacco.

273

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

274

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

275

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

276

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

277

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

278

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

279

protect cultivated crops are being investigated in laboratory studies.

280

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

281

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

282

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

283

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

284

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

285

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

286

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

287

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

289

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

290

291

292

293

294

Acknowledgements

295

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

296

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

297

and Dominique Marcon for technical assistance in photographic editing.

298

14

ACS Paragon Plus Environment

Page 14 of 27

Page 15 of 27

299

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Page 16 of 27

Page 17 of 27

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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.

378

18

ACS Paragon Plus Environment

Page 18 of 27

Page 19 of 27

379

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Page 20 of 27

Page 21 of 27

Journal of Agricultural and Food Chemistry

Fig 1

Methyl  gallate  

Ethyl  gallate  

ACS Paragon Plus Environment

Propyl  gallate  

Journal of Agricultural and Food Chemistry

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  

ACS Paragon Plus Environment

G  

H    

Page 23 of 27

Journal of Agricultural and Food Chemistry

Fig 3

CONTROL

1

1

2

2

3

CONTROL

3

4 SA 2mM

A

SA 2mM

4

5

5

6

6

B

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Fig 4

A

B

C

D ACS Paragon Plus Environment

Page 24 of 27

Page 25 of 27

Journal of Agricultural and Food Chemistry

c

Fig 5 b a a

A b c

a

a

B ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 27

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  

ACS Paragon Plus Environment

Page 27 of 27

Journal of Agricultural and Food Chemistry

TOC  

MG EG PG

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

ACS Paragon Plus Environment