Methyl jasmonate

Daidzein, daidzin, daidzin 6″-O-acetate, daidzin 6″-O-malonate, genistein, genistin, genistin 6″-O-acetate, genistin 6″-O-malonate, glycitein, glycitin, glycitin 6″-O-acetate, and glycitin 6″-O-malonate were purchased from FUJIFILM Wako Pure Chemical Corp (Osaka, Japan). CMS; dimethyl sulfoxide (DMSO); Gelrite™; indole-3-butyric acid (IBA); ascorbic acid; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; methyl jasmonate (MJ); and phosphate-buffered saline were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Mass-grade formic acid (FA), acetonitrile, methanol, and water were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The other chemicals employed in this study were of American Chemical Society grade or higher. The Sinhwakong soybean variety (an elite cultivar) was supplied by the National Institute of Crop Science (Cheonju, Republic of Korea).

Methyl jasmonate, 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS), HCl 37%, 2,4,6‐Tris (2‐pyridyl)‐s‐triazine (TPTZ), DPPH radical (diphenyl‐1‐picrylhydrazyl), (+)‐catechin, (−)‐epicatechin, quercetin 3‐O‐glucoside, and transresveratrol were obtained from Sigma‐Aldrich (US). L‐phenylalanine, 2‐methoxyphenol (Guaiacol), pyrocatechol, iron (III) chloride hexahydrate (FeCl₃ (6H₂O)), potassium persulfate (K₂S₂O₈), iron (II) sulfate heptahydrate (FeSO₄ (7H₂O)), sodium nitrite, Tween‐80, hydrogen peroxide 30%, Folin–Ciocalteu reagent, and polyvinylpolypyrrolidone (PVP) were purchased from Merck. Acetonitrile and rutin trihydrate were purchased from CHEMSOLUTE and Fluka, respectively. Trifluoroacetic acid (TFA) and methanol (all of HPLC grade) were obtained from VWR CHEMICALS. Milli‐Q water was acquired by SG water apparatus.

The seeds of vector control, LeMYC2overexpresser and LeMYC2 RNAitransgenic lines were sterilized with 4% sodium hypochlorite solution for 12 min and washed with sterile water for five times. The seeds were plated on to MS plates with 5 μM concentration of ABA (Sigma-Aldrich). The plates were kept in cold in dark for 4 days for stratification and then transferred to constant white light. The seeds were monitored for germination and greening from 3 days to 8 days. The seeds were counted as germinated when the radicle tip had fully expanded the seed coat. Control and different transgenic lines were germinated on MS with 10 μM of methyl jasmonate (Sigma) in magenta boxes, after stratification boxes were transferred to growth room and the root growth was monitored regularly and seedling were taken on plates and photographed and root length and lateral roots were measured.

Methanolic tomato extracts were analyzed by liquid chromatography coupled with mass spectrometry (HPLC-MS) in a Shimadzu apparatus equipped with an LC- 20AD pump and a CTO-10AS VP column oven coupled to a mass spectrometer with a simple quadrupole analyzer LCMS-2020 QP, with an electrospray ionization source (ESI). An ACE 3 C18 column (150 mm × 4.6 mm, 3 μm particle size) with an ACE3 C18 analytical pre-column was used for separation. The compounds were eluted with Methanol (LC-MS grade) (MeOH): MiliQ water with 1% acetic acid 5% MeOH for 5 min, followed by a gradient 5:100% MeOH for 30 min, 100% MeOH for 10 min and 100:5% MeOH for 8 min, with a flow rate of 0.5 ml/min. The nitrogen flow (drying gas for solvent evaporation) was 15 L/min. Electrospray capillary potential was + 4.50 kV and a Full Scan was used in positive mode (m/z 100–700) with a potential of 1.40 kV and a capillary temperature of 250°C. Stock solutions of extracts were injected at 0.25 mg/ml with a 5 μl injection through an automatic injector (SIL-20A XR). All extracts (0.25 μg/μl) were dissolved in 100% MeOH for injection. Pure compounds (lycopene, carotene, salicylic acid, chlorogenic acid, and methyl jasmonate from Sigma-Aldrich) were injected at 0.2 mg/ml and analyzed under the same conditions as described above.

The study was performed on two consecutive vintages (2019 and 2020) on 14-year-old Vitis vinifera L. cv Monastrell vineyards grafted onto 1103-Paulsen (clone 249) rootstock and trained in a bilateral cordon training system trellised to a three wire was used. Vine rows were arranged N-NW to S-SE with between-row and within-row spacing of 3 × 1.25 m and located in Cehegín (Murcia, Spain- latitude 38°6′39.35″, length 1°40′59.06″ and altitude of 433 m). The vineyard was drip-irrigated and liquid organic matter was added as a fertilizer. The experiments were conducted in a randomized block design, in which all treatments were applied to three replicates, using 10 vines for each replication. Two foliar treatments were applied to the plants in spray form as a water suspension of MeJ 10 mM (methyl jasmonate) (Sigma Aldrich, St. Louis, MO, USA) and a water suspension of MeJ-ACP 1 mM (Mej-doped calcium phosphate nanoparticles) at veraison. Approximately 200 mL of the product was applied to each plant prepared with Tween 80 (Sigma Aldrich, St. Louis, MO, USA) as the wetting agent (0.1% v/v). Control plants were sprayed with aqueous solution of Tween 80 alone. For all treatments, a second application was performed 7 days after the first.

The pure compounds cis‐jasmone and methyl jasmonate were purchased from Sigma‐Aldrich, and allylanisole from Merck (Darmstadt, Germany). All pure compounds were blended at a ratio of 1:1 (wt/wt), i.e., each compound represents half of the total dose tested. Pure compounds as well as their three blends were diluted in pure ethanol (Merck) at a ratio of 1:10 by volume. Subsequently, the respective amount of distilled water plus Triton X‐100 (0.05%; Sigma‐Aldrich) as surfactant was added to obtain a range of concentrations (0.1–2%). The control solution consisted of ethanol and distilled water with the surfactant at a ratio of 1:10.