# VOCs Are Relevant Biomarkers of Elicitor-Induced Defences in Grapevine
## 4 Materials and Methods
### Stilbene Analysis  
Leaves were harvested at five times points from D1 to D8 (Figure 6A). They were ground in liquid nitrogen to a fine powder that was freeze-dried, and 100 mg were extracted with 8 mL of methanol overnight at 4 \({}^{\circ}\)C. Six mL of supernatant were collected, evaporated, and the pellet was dissolved in 1 mL of methanol/water (70/30, _v/v_). The leaf extracts were purified to remove chlorophylls on a Supelclean LC-18 solid phase extraction column (Supelco Inc., Bellefonte, PA, USA). Eluates obtained with methanol/water (90/10, _v/v_) were evaporated and resuspended in 600 uL of methanol/water (50/50, _v/v_). The phenolic extracts were diluted, centrifuged at 13,500 rpm for 10 min, and stored at \(-20\)\({}^{\circ}\)C until use.  
Stilbenes were analysed by a multiple reaction monitoring (MRM) mode HPLC-MS/MS methodology adapted from [PERSON], et al. [52]. The device consisted of a 1260 Infinity UPLC system (Agilent Technologies, Courtaboeuf, France) coupled to a 6430 triple quadrupole mass spectrometer (Agilent Technologies, Les Ulis, France). Four microlitres of leaf extract were injected into an Agilent Poroshell 120 EC-C18 column (150 mm \(\times\) 2.1 mm, 2.7 um diameter) thermostated at 30 \({}^{\circ}\)C. A flow rate of 0.3 mL/min of solvent A (99.9% distilled water, 0.1% formic acid) and solvent B (99.9% acetonitrile, 0.1% formic acid) was used with the following gradient: 0-5 min, 5-18% B; 5-15 min, 18-43% B; 15-17 min, 43-95% B; 17-20 min, 95% B; 20-22 min, 95-5% B. Acquisitions were done in the negative mode with the following source parameters: gas temperature 350 \({}^{\circ}\)C, voltage 3000 V, gas flow 1 L/min, nebuliser pressure 15 psi. Calibration curves (concentrations ranging from 0.004 to 10 mg/L) of pure standards (_trans_-resveratrol and _trans_-plecial, 3 replicates) were established to determine the stilbene concentrations in mg/g dry weight (DW). Both standard stilbenes were produced and purified in laboratory conditions (Oenology Research unit, Bordeaux university, Villenave d'Ornon, France). _cis_-resveratrol and _cis_-plecial were expressed as their corresponding _trans_-isomers. Supplemental Table S3 describes the detection method and quantification parameters.  
Supplementary MaterialsThe following are available online. Figure S1: Time-dependent VOC emission. Table S1: VOCs induced by Bastid(r), chitosan, an oligogalacturonide (OG), and Bion(r). Table S2: Parameters of the VOC calibration standards. Table S3: Parameters of the stilbene analysis method.  
**Funding:** This work was supported by the French Ministry of Agriculture [CASDAR-CODEVI project], the Conseil Regional de Bourgogne-Franche-Comte (France), the Bordeaux Metabolome Facility and MetaboHUB (ANR-11-INBS-0010 project).  
**Data Availability Statement:** Analyzed data are provided in the table of the article.  
**Acknowledgments:** All co-authors are very thankful to Goemar company for kindly providing elicitors (PS3 and OG). We also thank the INRAE greenhouse team for their help in producing grapevines. We gratefully acknowledge [PERSON] for correcting the English language of the manuscript.  
**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.  
**Sample Availability:** No available samples.