Milli q element water purification system

Ultrapure water (resistivity ≥18.2 MΩ cm) was obtained from a Milli-Q Element water purification system (Millipore, Guyancourt, France). Trace metal analysis grade 14 M HNO₃ and 12 M HCl were acquired from PrimarPlus (Fisher Chemicals, Loughborough, UK), and a Savillex DST-4000 acid purification system (Savillex Corporation, Minnetonka, MN, USA) was used for further purification. TraceSELECT^® 9.8 M H₂O₂ was acquired from Sigma-Aldrich (Overijse, Belgium). Isotopic reference material NIST SRM 976 (National Institute of Standards and Technology—NIST, Gaithersburg, MD, USA) was used as an external standard in the Cu isotope ratio measurements. Ga and Cu single-element standard stock solutions (Inorganic Ventures, Nieuwegein, The Netherlands) were used as admixed internal standard and in-house standard solution for quality control of the isotope ratio measurements, respectively. Sample preparation was performed in a class-10 clean lab (PicoTrace, Göttingen, Germany).

Trace metal analysis grade 14 M HNO₃ and 12 M HCl acquired from PrimarPlus (Fisher Chemicals, UK) were further purified via sub-boiling distillation in a Savillex® DST-4000 acid purification system (Savillex Corporation, United States). TraceSELECT® 9.8 M H₂O₂ was acquired from Sigma-Aldrich (Belgium). Ultrapure water with a resistivity ≥18.2 MΩ cm was obtained from a Milli-Q Element water purification system (Millipore, France) and will further be referred to as MQ-water. The isotopic reference materials used for the isotope ratio measurements were NIST SRM 976 for Cu (National Institute of Standards and Technology–NIST, United States) and IRMM-014 for Fe (Institute for Reference Materials and Measurements–IRMM, Belgium). Adequate dilutions and mixtures of 1 g.L⁻¹ single-element standard stock solutions of Cu, Fe, Ni and Ga (Inorganic Ventures, the Netherlands) were used for quantification, mass bias correction (Ni and Ga as admixed internal standards), and for checking the quality of the isotope ratio measurements (Cu and Fe in-house standards). Sample preparation was performed in a class-10 clean lab (PicoTrace, Germany).

Acetonitrile, formic acid, and methanol were purchased from Fisher Company (Fairlawn, NJ, USA). D-(+)-dextronic acid δ-lactone was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Cleanert^® PEP-SPE resin (150 mg/6 mL) was obtained from Bonna-Agela (Wilmington, DE, USA).
The standards (purities > 95%) used for identification and quantification of the volatile compounds 1-hexanol, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, (E)-3-hexen-1-ol, (Z)-3-hexen-1-ol, hexanal, (E)-2-hexenal, 1-nonanol, furfural, hexanoic acid, limonene, D-limonene, myrcene, linalol, α-terpineol, 4-terpineol, β-citronellol, citral, geranylacetone, geraniol, nerol, cis-furan oxidin, spirulina, (Z)-orange sterol, (E)-β-damascenone, (E)-β-ionone, and 4-methyl-2-pentanol were all supplied by Sigma-Aldrich (St. Louis, MO, USA). The deionized water (<18 WΩ resistance) was obtained through a Milli-Q Element water purification system (Millipore, Bedford, MA, USA). All the other chemicals or reactives without especial mention were purchased from Beijing Chemical Works (Beijing, China).

The malic acid contents in the apple fruits were measured using High Performance Liquid Chromatography (HPLC) as described in our previous protocol [34 (link)]. Briefly, approximately 5 g of frozen powder was dissolved in 20 mL of ddH₂O, which was generated by a Milli-Q Element water purification system (Millipore, Bed ford, MA, USA). The mixture was put into an ultrasonic bath and maintained at room temperature for 30 min under continuous ultrasonic shaking. The mixture was then centrifuged at 1200 rpm for 15 min at 4 °C The supernatants were decanted and filtered through a 0.22 μm Sep-Pak filter (Anpel, Shanghai, China). The filtered fluid was used to measure the malic acid content via an Agilent 1260 Infinity HPLC system (Milford, MA, USA) coupled to a diode array detector at 210 nm. Chromatographic separation was performed on an Athena C18 column (1000 nm, 4.6 nm × 250 nm, 5 μm); the column temperature was maintained at 40 °C The mobile phase was a 0.02 mol/L KH₂PO₄ solution at a pH of 2.4, and the flow rate was 0.8 mL/min. Three biological replicates were conducted. The malic acid standard used was purchased from Sigma (St. Louis, MO, USA), and dissolved in deionized water.