Analysis of Phytohormones and Sugars in Leaves

Phytohormones and sugars were analyzed from 10 mg freeze-dried, ground leaf material of the photosynthesis experiment (n = 6). Phytohormone analysis was carried out on an LC/MS/MS system as previously described (Eberl et al., 2018 (link)). Data were processed using ANALYST 1.5.2 (AB Sciex, Framingham, MA, United States) and hormones were quantified relative to the peak area of their corresponding standard (D₄-salicylic acid and D₆-abscisic acid; Santa Cruz Biotechnology, Dallas, TX, United States). For sugar analysis, extracts were diluted 1:10 with water prior to analysis on an Agilent 1200 HPLC system (Agilent, Santa Clara, CA, United States) coupled to an API 3200 tandem mass spectrometer (AB Sciex). The analytes were separated on an hydrophobic interaction liquid chromatography (HILIC)-column (apHera NH₂ Polymer; 15 × 4.6 mm, 5 μm; Supelco, Bellefonte, PA, United States) with a water/acetonitrile gradient (flow, 1.0 ml min^-1), for more details see Madsen et al. (2015) (link). The data were processed using ANALYST 1.5.2 (AB Sciex) and the compounds were quantified using an external standard curve. For this, a mixture of glucose, fructose and sucrose (Sigma-Aldrich, St. Louis, MO, United States) was analyzed at six different concentrations ranging from 20 to 1.25 μg ml^-1.