Phylloquinone

For tocopherol and phylloquinone analyses, 0.1g freeze-dried powder derived from leaves of 3-month-old plants was added into 3ml CHCl₃/n-hexane (3:7, v/v) solvent under dim light and ultrasonically oscillated for 10min. After centrifugation at 5000g for 8min, the clear supernatant was taken and the extraction was repeated twice more. The resulting supernatant was evaporated to dryness under nitrogen and dissolved in 1.5ml CHCl₃/methanol (3:7, v/v). 30μl of the sample solution was used for reverse phase-HPLC analysis. All the standards of α-, γ-, δ-tocopherol and phylloquinone were purchased from Sigma-Aldrich (Shanghai, China). Target analytes were separated on a YMC Carotenoid C30 Column (250×4.6mm, 5μm) under the following condition: 97% methanol for 6min, 97%-100% methanol in 10min, 100% methanol for 20min, 100%-97% methanol in 2min, and re-equilibration at 97% methanol for 6min at a flow rate of 0.8ml/min. Analytes were detected by photodiode array detector (PDA) at 292nm (S4 Fig). Ascorbate content was measured by means of dichloroindophenol titration, according to the method described in [48 (link)]. Total carotenoid and chlorophyll contents were measured with spectrophotometry, according to the method described in [49 ].

Tissues used in this study were collected from 1-year-old (leaves, stems, bark, and roots) and mature elite (flowers and hulls) black walnut trees located at Martell Forest (West Lafayette, IN, USA). Naphthoquinone standards of juglone, phylloquinone, menaquinone-4 (MK-4), and plumbagin were from Sigma-Aldrich. Unless otherwise mentioned, all other reagents were from Fisher Scientific. Ultrapure water and high-performance liquid chromatographic (HPLC)- or gas chromatographic (GC)-grade solvents were used for all metabolite extractions. HPLC analyses were carried out on an Agilent 1260 Infinity series instrument (Agilent Technologies) equipped with diode array and fluorometer detection modules employing Chemstation software. An Agilent 7890B GC coupled with a 5977A mass spectrometer (MS) employing Chemstation software was used to perform GC-MS analyses.