Quantification of simple sugars
Using 25 mg of freeze-dried leaf tissue, soluble sugars were extracted and analyzed by the method described here (detailed method to be published elsewhere). Briefly, soluble sugars were extracted in 80% EtOH at 65C for 30 minutes. The extract was then filtered using 0.45 μm nylon syringe filter. The sugar profiles were determined using reverse-phase high performance liquid chromatography paired with a refractive index detector (HPLC-RID, Shimadzu Scientific Instruments Inc, Columbia MD). For sugar separation, an isocratic mobile phase of 80% acetonitrile at a 2 ml min-1 flow rate and a Luna NH2 column (250×4.6 mm, 5 μm, Phenomenex Inc, Torrance, CA) was used. Each sugar was quantified using a 6-point external standard curve (0.0625- 2 mg ml-1). The chromatographs were analyzed, and the data were processed using Perkin Elmer TotalChrom software (version 6.2.1). To quantitate those sugars which did not separate, the areas and concentrations of each were added together to create a combined standard curve (i.e. xylose+arabinose, glucose+galactose, maltose+trehalose).