Jasmonic acid

Rosettes of seven-week-old plants were frozen immediately after harvest and ground in liquid nitrogen. Four pools of 6 rosettes were collected. Frozen material (100 mg) was extracted with 3 ml of acetone/water/acetic acid (80/19/1, v:v:v) containing the following stable isotope labelled internal standards: 10 ng [4-2H] ABA (NRC-CNRC Plant Biotechnology Institute, Saskatoon, Canada), 50 ng [4-2H] salicylic acid (Olchemlm, Olomouc, Czech Republic), 1 ng [5-2H] jasmonic acid (CDN Isotopes CIL Cluzeau, Sainte Foy la Grande, France), and 10 ng [6-13C] indole-3-acetic acid (Cambridge Isotope Laboratory, Andover, MA). The extract was vigorously shaken for 30 s, sonicated for 1 min at 25 Hz, shaken for 10 min at room temperature, and then centrifuged (8230 g, 4°C, 15 min). The supernatants were collected and the pellets were extracted again with 1 ml of the same extraction solution, and then vigorously shaken (1 min) and sonicated (1 min, 25 Hz). Following centrifugation, the two supernatants were pooled and dried. The dry extract was dissolved in 140 µl acetonitrile/water (50/50, v/v), filtered, and submitted to analysis by HPLC-electrospray ionisation-MS/MS (HPLC-ESI-MS/MS). The compounds were introduced into the ESI source using a Waters 2695 separation module (Alliance; Waters, Milford, MA, USA) equipped with a Waters 2487 dual UV detector. Separation was achieved on a reverse-phase column (Uptisphere C18 ODB, 150*2.1 mm, Interchim, Montluçon, France), using a flow rate of 0.15 ml min⁻¹ and a binary gradient as follows: (A) acetic acid 0.1% (v/v) and (B) acetonitrile. The solvent gradient was programmed as follows: 0–5 min, 20% A; 5–15 min, 50% A; 15–30 min, 100% B; and 30–42 min, 20%. The analyses were performed on a Waters Quattro LC Triple Quadripole Mass Spectrometer (Waters) operating in a Multiple Reaction Monitoring (MRM) scanning mode. The instrumental parameters were set as follows: capillary, 2.70 kV (negative mode); extractor, 3 V; and source block and desolvation gas temperatures, 120°C and 350°C, respectively. Nitrogen was used for the nebulization and desolvation (77 L h⁻¹ and 365 L h⁻¹, respectively), and argon was used as the collision gas at 2.83 10⁻³ mbar. For a 5-µL injection volume of sample prepared and reconstituted in 140 µl of 50/50 acetonitrile/H2O (v/v), the limit of detection (LOD) and limit of quantification (LOQ) were extrapolated for each hormone from a calibration curve and sample using the Quantify module of MassLynx (version 4.1 software). The parameters used for MRM quantification and the LOD and LOQ are listed in Tables S4 and S5, respectively. The amount of JA was expressed as a ratio of peak areas (209>62/214>62) per fresh weight, due to impurities contained in the D5-JA standard.

Mass spectrometry-based metabolomics profiling was performed on frozen prostate cell pellets (∼10 million cells). The process of metabolite extraction involved introduction of equimolar mixture of 11 standard compounds dissolved in methanol (Epibrassinolide, [D₃] Testosterone, [¹⁵N] Anthranilic acid, Zeatine, Jasmonic acid, Gibberelic acid, [D₄] Estrone, [¹⁵N]-Tryptophan, [D₄] Thymine, [¹³C] Creatinine and [¹⁵N] Arginine) followed by homogenization of the cells. The homogenate was then subjected to extraction with sequential use of aqueous (chilled water) and organic (chilled methanol and chloroform) solvents in the following ratio 1∶4∶3∶1 (water∶methanol∶chloroform∶water) [28] . The resulting extracts were de-proteinized using a 3 KDa molecular filter (Amicon Ultracel -3K Membrane, Millipore Corporation, Billerica, MA) and the filtrate containing metabolites were dried under vacuum (Genevac EZ-2^plus, Gardiner, NY). Prior to mass spectrometry analysis, the dried extract was resuspended in identical volume of injection solvent composed of water∶methanol (50∶50) with 0.2% acetic acid and subjected to liquid chromatography (LC) mass spectrometry. As additional controls to monitor the profiling process, an equimolar mixture of 11 standard compounds (Epibrassinolide, [D₃] Testosterone, [¹⁵N] Anthranilic acid, Zeatine, Jasmonic acid, Gibberelic acid, [D₄] Estrone, (¹⁵N) Tryptophan, [D₄] Thymine, [¹³C] Creatinine, and [¹⁵N] Arginine) and a characterized pool of mouse liver (extracted in tandem with cell lines) were analyzed along with the cell line samples. Each of these controls, were included multiple times into the randomization scheme such that samples preparation and analytical variability could be constantly monitored. Furthermore, analysis of each cell line was succeeded by at least two blank runs, to prevent any carryover of metabolites between samples. Figure S1 illustrates the reproducibility in the profiling process monitored using the standard mixture described above. Notably the CV for the entire profiling process measured using five independent replicates of the mouse liver extract mentioned above was less than 5%.

Fresh plant material (1g) was homogenized in 100 mM Tris-HCl (pH 7.5) in presence of DTT (Dithiothreitol, 5 mM), MgCl₂ 10 mM, Ethylenediaminetetraacetic acid (EDTA, 1 mM), magnesium acetate 5 mM, Polyvinylpyrolidone (PVP-40 1.5%), phenylmethanesulfonyl fluoride (PMSF 1 mM) and aproptinin 1 μgmL^-1. After the filtration, the homogenate was centrifuged at 10,000 rpm for 15 min. The supernatant collected after centrifugation served as enzyme source. For the analysis of APX activity, tissues were separately homogenized with 2 mM AsA. All experiments were performed at 4°C.
Activity of SOD was estimated according to Kono (1978) (link) following the photo reduction of nitroblue tetrazolium (NBT). The absorbance was recorded spectrophotometerically (Beckman 640 D, USA) at 540 nm. SOD unit is the quantity of enzyme that hamper 50% photoreduction of NBT and is expressed as EU mg^-1 protein.
The activity of POD was estimated according to the method proposed by Putter and Becker (1974) . The rate of production of oxidized guaiacol was estimated spectrophotometerically (Beckman 640 D, USA) at 436 nm. The activity of POD was expressed as EU mg^-1 protein.
Catalase activity was estimated by the method of Aebi (1984) (link). The OD was taken spectrophotometerically (Beckman 640 D, USA) at 240 nm and the activity was expressed as EU mg^-1 protein.
For the determination of APX activity, the procedure of Nakano and Asada (1981) was used. The OD was recorded at 265 nm by spectrophotometer (Beckman 640 D, USA) and the activity was expressed as EU mg^-l protein.

We collected female flowers from all the treated and untreated plants to evaluate the difference in B foliar spray on JA content. JA content was determined with a triple quadrupole liquid chromatography-mass spectrophotometry system following the method described by Wang et al. [6 (link)] for sample preparation and analysis. JA content was visualized as mean ± standard in a bar diagram. The significant difference was calculated using the student’s t-test between boron untreated and treated samples.

The content of melatonin and jasmonic acid was determined using the double antibody sandwich method. Extract fruit pulp (0.25 g) using 2.25 mL phosphate buffer (pH 7.4). Purified plant jasmonic acid (JA) or melatonin (MT) antibodies are coated on a microplate to prepare solid-phase antibodies. Jasmonic acid (JA) or melatonin (MT) is sequentially added to the microplate of the coated monoclonal antibody, and then combined with Horseradish Peroxidase labeled jasmonic acid (JA) or melatonin (MT) antibodies. After thorough washing, substrate is added for color development. Measure the absorbance (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay kit (Tongwei Biotechnology, Shanghai, China) to detect endogenous melatonin and JA.