Optima 3000

Faeces and urine of Studies 1, 3 and 4 were analysed as described previously (except neutral sterols and bile acids) [11 (link),19 (link),20 (link)]. In Study 2, mineral concentrations in faeces were determined following pressure digestion using ICP-OES (Optima 3000, Perkin Elmer, Waltham, MA, USA).
Faecal neutral sterols (cholesterol and its metabolites coprostanol, coprostanone, cholestanol, cholestanone, and cholestenone) and bile acids (iso-lithocholic acid: iLCA, lithocholic acid: LCA, iso-deoxycholic acid: iDCA, deoxycholic acid: DCA, cholic acid: CA, chenodeoxycholic acid: CDCA, and 12keto deoxycholic acid: 12keto DCA) were analysed as described previously [22 (link)]. Briefly, neutral sterols were extracted with cyclohexane following a mild alkaline hydrolysis. Analysis was performed by GC-FID (GC17A-AF Vers. 3, Shimadzu, Kyoto, Japan). Bile acids were extracted with diethyl ether following a strong alkaline hydrolysis. Thereafter, extracts were methylated, silylated, and analysed by GC-MS (GC17-QP5000, Shimadzu, Kyoto, Japan).

At the end of the experimental period, the olive shoots were collected, and the leaves from the different treatments were first washed in distilled water. This washing liquid was analysed by ICP-OES and liquid chromatography, as described below, for determining the leaf surface K and anion concentrations.
For tissue elemental analysis, pre-washed olive leaves (i.e., after the preliminary water described above) were subsequently washed in a 0.1% detergent (Fairy, P&G), acidulated solution (0.1 N HCl). The plant tissues were then rinsed twice in tap water, and then in distilled water, and were consequently oven-dried at 70 °C for two days, then weighed and ground prior to mineral element determination after dry-ashing. The concentration of nutrients in the plant tissues was detected by inductively coupled plasma (ICP, PerkinElmer, Optima 3000), following the UNE-EN ISO/IEC 17025 standards for calibration and testing laboratories (CEBAS-CSIC Analysis Service, Murcia, Spain). On the other hand, the anions were analysed by liquid chromatography (CEBAS-CSIC Analysis Service, Murcia, Spain), after the extraction of approx. 0.3 g dry weight (D.W.) of leaf tissue in distilled water for 2 h, and centrifugation and filtration with 0.45 µm filters.

The detection of metals in the apo-and metal-incubated forms of purified EfeM_Psy was performed by ICP-OES, using the instrument PERKIN-ELMER Optima 3000. The metal stock solutions were prepared by dissolving the chloride salts of each metal tested in 30 mM MES, pH 6.0. In the case of ferrous iron, the stock solutions were prepared in 30 mM MES, pH 5.0, to reduce oxidation of the ferrous iron. The proteins (2–50 µM) were incubated with 0.25 mM metal stock solution separately (Cu²⁺, Zn²⁺, Fe²⁺, Mg²⁺, Mn²⁺ and Fe³⁺) at 4 °C for 1 h. The unbound metals were then removed by subjecting the incubated samples to dialysis, using 12 kDa MWCO dialysis tubing against metal-free buffer (30 mM MES, pH 6.0 or pH 5.0) with the replacement of buffer every 8 h three to four times to remove the unbound metals. In the case of the Fe²⁺ binding experiments, to prevent oxidation the buffer pH was maintained at 5.0 and nitrogen gas was bubbled/purged into the dialysis system at regular intervals.

Metallic elements were determined using the PerkinElmer Optima 3000 emission spectrometer that can measure wavelengths between 167–782 nm for over 5000 emissions. The liquid sample was rapidly atomized using an argon plasma at 7000 °C and electrons excited for detection and analysis using WinLab32 software. The non-metallic elements were analysed using the Vario EL Elementar equipment. It was conducted in accordance to the Petrochemical industry standard GB/T 19143-2003.

For an ionic analysis, the leaves were washed well with distilled water and lyophilized. Finely ground samples of lyophilized leaves were digested in a microwave oven (CEM Mars Xpress, Matthews, NC, United States) by HNO3:HClO4 (2:1) digestion. The elements were detected by an inductively coupled plasma (ICP) analysis (Optima 3000, PerkinElmer, Norwalk, CT, United States).