Quantifying Leaf Metabolites in Plants

For determining the content of titratable acids, aliquots of methanol extracts were titrated against NaOH (0.05 mm) to a neutral endpoint and expressed as millimoles of H⁺ per gram fresh weight (fwt), as described by Cushman et al. (2008a (link)). Starch was extracted from the leaf mesophyll as described previously (Haider et al., 2012 (link)), and its content in mesophyll was measured as glucose equivalents using the colorimetric phenol/sulphuric acid test described by DuBois et al. (1956) . To determine starch content in guard cells, the peels were fixed (50 % v/v methanol, 10 % v/v acetic acid) and stained with Lugol’s iodine solution as described by Horrer et al. (2016) (link). Soluble sugars were measured in both tissues using the high-pressure ion chromatography (HPIC) technique (Thermo Scientific Dionex), and the amount of sugars (in micromoles per gram fresh weight) was calculated based upon standards of glucose, fructose, sucrose and maltose. Malate content in both mesophyll and guard cell-enriched epidermis was determined by the enzymatic method developed by Hohorst (1970) .