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 60C 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.