2.5 Metabolite analyses
Non-structural carbohydrates (NSC) in leaves and bark are predominately present as starch (St) and soluble sugars (Ss) (Hartmann & Trumbore 2016). To determine if girdling affected the concentration of St and/or Ss, we collected three fully expanded leaves from the upper and lower crown of each tree immediately before girdling (10:00 h, 16 May) and 9 days after girdling at the end of the experiment (10:00 h, 25 May). No branches were present below the girdle, thus leaves sampled from the lower crown originating from immediately above the girdle. Bark tissue (3 cm2) was sampled at the same time from the main stem close to the installation sites of sap flow sensors and dendrometers at the base and top of stems. Immediately after collection, samples were snap-frozen in liquid N and stored at -80 °C prior to further processing.
Prior to extractions, leaves and bark samples collected prior and during the girdling experiment were dried at 70 °C to a constant weight and ground to a fine powder using a ball mill. Concentration of St and Ss was determined using a procedure adapted from Tissue and Wright (1995) that required 15-20 mg of sample material. In a first step, Ss were washed out of solids using a methanol-chloroform-water solution (12:5:3 v/v). In the following step, St was extracted from solids using perchloric acid (35% v/v). Concentrated sulphuric acid was then used to hydrolyse both Ss and St to glucose, before addition of phenol induced a colorimetric reaction. Finally, absorbance of glucose-phenol solutions was measured at 490 nm in a photometer (DU 800 Spectrophotometer, Beckman Coulter, Sydney, Australia), and readings were converted to quantities of Ss and St using a simple mathematical approach.