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 65◦C 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).