Plant protection based on novel alternative strategies is a major concern in agriculture to sustain pest management. The marc extract of red grape cultivars reveals plant defence inducer properties. Treatment with grape marc extract efficiently induced hypersensitive reaction-like lesions with cell death evidenced by Evans Blue staining of tobacco leaves. Examination of the infiltration zone and the surrounding areas under UV light revealed the accumulation of autofluorescent compounds. Both leaf infiltration and a foliar spray of the red grape extract on tobacco leaves induced defence gene expression. The PR1 and PR2 target genes were upregulated locally and systemically in tobacco plants following grape marc extract treatment. The grape extract elicited an array of plant defence responses making this natural compound a potential phytosanitary product with a challenging issue and a rather attractive option for sustainable agriculture and environmentally friendly practices.

To cite this version:Razik Benouaret, Eric Goujon, Pascale Goupil. Grape marc extract causes early perception events, defence reactions and hypersensitive response in cultured tobacco cells. Plant Physiology and Biochemistry, Elsevier, 2014Elsevier, , 77, pp.84-89. <10.1016Elsevier, /j.plaphy.2014.01.021>. Author manuscript, published in Plant Physiology and Biochemistry (2014) 77: 84-89 DOI:10.1016/j.plaphy.2014 1 -A polyphenol-rich extract from grapes induced chemoperception in tobacco suspension cells -GME induced modification of plasma membrane properties -GME elicits defence genes in tobacco cells -GME-induced cell death was mediated by proteases and de novo protein synthesis -This study evidences the ability of GME to induce hypersensitive response Graphical abstractHighlights Abstract: Grape marc extract (GME) showed elicitor activity on suspension-cultured cells of tobacco. The BY-2 cells reacted to GME (0.25% and 0.125%) with a long-sustained pH rise in their growth medium. Using EGTA or LaCl 3 , we showed that extracellular alkalinization depended on Ca 2+ mobilization. The tobacco BY-2 cells challenged with GME promoted cell death and the upregulation of defence-related genes such as PR3, PAL and CCoAOMT. Cell death rate was quantified using an experimental calibrated Evans Blue assay. The GMEinduced cell death was dose-dependent and occurred in 24 h. Longer exposure increased the extent of tobacco cell death. To investigate a potential hypersensitive reaction, we tested the effect of various inhibitors of protein synthesis (cycloheximide) and proteases (aprotinin, pepstatin and E-64) on GME-induced cell death. All these chemicals reduced GME-induced cell death rate in 30 min. Overall, our findings indicate that GME elicits early perception events, defence reactions and cell death requiring protein synthesis and proteases.

Alkyl gallates showed elicitor activities on tobacco in both whole plants and cell suspensions. Methyl gallate (MG), ethyl gallate (EG), and propyl gallate (PG) infiltration into tobacco leaves induced hypersensitive reaction-like lesions and topical production of autofluorescent compounds revealed under UV light. When sprayed on tobacco plants at 5 mM, EG promoted upregulation of defense-related genes such as the antimicrobial PR1, β-1,3-glucanase PR2, Chitinase PR3, and osmotin PR5 target genes. Tobacco BY-2 cells challenged with EG underwent cell death in 48 h, which was significantly reduced in the presence of the protease inhibitor aprotinin. The three alkyl gallates all caused alkalinization of the BY-2 extracellular medium, whereas gallic acid did not trigger any pH variation. Using EGTA or LaCl, we showed that Ca mobilization occurred in BY-2 cells elicited with EG. Overall, our findings are the first evidence of alkyl gallate elicitor properties with early perception events on the plasma membrane, potential hypersensitive reactions, and PR-related downstream defense responses in tobacco.