Gallery plus automated photometric analyzer

Each July, samples of about 100 fully expanded leaves from the current season’s growth of shoots without fruit (diagnostic leaves) were collected from each measured tree. The leaf samples were rinsed in deionised water, oven-dried (70 °C), ground to a powder, and 0.1 g were digested with sulfuric acid and hydrogen peroxide (Merck, Darmstadt, Germany) [67 ]. The P and N content was determined by colorimetric analysis (Gallery Plus Automated Photometric Analyzer, Thermo Scientific, Waltham, MA, USA). The K content was determined using an atomic absorption spectrometer (AAnalyst 200, Perkin-Elmer, Waltham, MA, USA). Fruit samples collected at harvest were crushed to paste and handled similar to the leaf samples.

BLA activity was measured as previously41 (link). 2.0 mL sample cups (Kartell Labware) were pre-blocked using 50 mM NaH₂PO₄ pH 7.0 with 3% BSA at 37 °C for 2 hours. The samples cups were then rinsed twice using 50 mM NaH₂PO₄ pH 7.0. Enzymes were diluted in the sample cup to a concentration of 0.25 nM using 50 mM NaH₂PO₄ pH 7.0 with 0.1% BSA. Samples were then allowed to react in 50 mM NaH₂PO₄ pH 7.0 with 100, 50, 30, 20, 15, or 10 μM Nitrocefin (Oxoid) in a Gallery Plus Automated Photometric Analyzer (Thermo Scientific) at 30 °C. Readings were taken every 9 seconds for a period of 99 seconds at a wavelength of 500 nm (Δε₅₀₀ = 15,900 M⁻¹cm⁻¹)42 (link). The first 6 data points were linear and were used to obtain the initial rates of reaction. A Lineweaver-Burk plot was used to extrapolate the kinetic parameters. Errors for kinetic values were obtained by calculating the kinetics for 3 separate sets of data and then calculating the average and standard deviation of the 3 kinetic values.