Quantification of free amino acids, free polyamines, and soluble
inorganic ions
Freeze-dried leaf tissue (25 mg) was subjected to 3 freeze-thaw cycles
in 5% perchloric acid (PCA). PAs and AAs were simultaneously dansylated
and quantified via reverse-phase HPLC per Minocha & Long (2004) with
minor modifications described in (Majumdar et al. 2018). Briefly,
PCA extracts were incubated at 60○C for 30 min
containing 2.694 M sodium carbonate solution and dansyl chloride
(dissolved in HPLC grade acetone). After the incubation period, the
reaction was terminated using acetic acid. Sample tubes were kept open
under a flow hood to allow CO2 to escape. The acetone
was then removed under vacuum. Filtered HPLC-grade methanol was used to
resuspend the dansylated compounds. The solution was then filtered using
0.45 μm nylon syringe filter. Gradient elution of mobile phase A
(acetonitrile) and mobile phase B (25mM sodium acetate buffer (pH 5.94)
containing 3% 1-propanol and 10% acetonitrile) was used. PAs and AAs
were analyzed, and the data were processed using Perkin Elmer TotalChrom
software (version 6.2.1). PAs were quantified using an internal
standard, AAs with external standard curves. To quantitate those AAs
which did not separate, the areas and concentrations of each were added
together to create a combined standard curve (i.e. Arg+Thr,
Arg+Thr+Gly).
The 5% PCA extracts analyzed for AAs and PAs were also analyzed for
inorganic ions and P via simultaneous axial inductively coupled plasma
optical emission spectrophotometer (ICP-OES, Vista CCD, Varian, Palo
Alto, CA). The extract was diluted 100x in ddH2O. For
each sample, triplicate readings were taken, and the spectral data were
analyzed with Vista Pro software (version 4.0) using a set of 6
standards of the elements of interest. ICP analysis was done in
accordance with EPA SW-846 compendium, method 6010.