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1995-2009 Foliar analyses

Extractions: Soluble ions, free PAs and free AAs were extracted from the foliar and sapwood plug samples collected in 5% perchloric acid (PCA) by three freezing and thawing cycles according to procedures described previously (Minocha et al. 2000; Minocha et al. 1994). All samples were stored at -20 ºC until analysis. Separate analyses were performed for each individual tree sample.

Polyamines and Amino Acids

Polyamines (1998-2004): Perchloric acid extracts were dansylated and quantified according to the procedure described in Minocha et al. (1990). A PerkinElmer (Norwalk, CT, USA) HPLC system consisting of Series 200 pump, autosampler and, LC 240 fluorescence detector with a 20 µl loop (10 µl injection volume) was used for quantification. A Pecosphere – 3 x 3 CR C18, 3 μm, 33 x 4.6 mm I.D. analytical column was used for the separation of PAs. Excitation and emission wavelengths were set at 340 and 510 nm, respectively. A Gilson (Middleton, WI, USA) 712 HPLC System Controller (version 1.2) was used to integrate the data.

Amino Acids (1999-2000): Amino acids data were collected starting in 1999 using PCA extracts that were dansylated and quantified according to the procedure described in Minocha and Long (2004) with a PerkinElmer Pecosphere Reduced Activity RP C18, 3µm, 100 x 4.6 mm analytical column.

Simultaneous polyamines and amino acids processing and quantification (2004-2018): Both PAs and AAs were simultaneously dansylated and quantified within a single run using the method described in Minocha and Long (2004b). The dansylation reaction was terminated in these samples using L-asparagine (50 µL of 20 mg mL-1 in water) rather than Alanine (Ala), described in the original method. A new series 200 PerkinElmer HPLC system fitted with a Phenomenex (Torrance, CA, USA) SynergiTM Hydro-RP, 4 µm, 100 x 4.6 mm I.D. analytical column, a C18 SecurityguardTM, 5 µm, 4 x 3 mm I.D. cartridge guard column, and a PerkinElmer C18 Scavenger, 10 µm, 33 x 4.6 mm I.D. cartridge column was used. Excitation and emission wavelengths were set at 340 and 515 nm respectively. Data were integrated using TotalChrom HPLC software package (PerkinElmer, version 6.2.1).

Exchangeable Inorganic ions (soluble in 5% PCA)

Exchangeable Inorganic ions (1995-1998). The quantitation of soluble inorganic ions of Ca, Mg, Mn, K, Al and P (defined as the fraction of total ions within cells that is extractable in 5% PCA) was conducted using Beckman Spectrospan V ARL DCP (Direct Current Plasma Emission Spectrometer, Beckman Instruments, Inc., Fullerton, CA) using the Environmental Protection Agency’s method number 66-AE0029 (1986). Supernatants of 3x frozen and thawed samples in PCA were diluted (foliage 100x, sapwood plugs 50x) with distilled deionized water for estimation of soluble ions.

Exchangeable Inorganic ions (1999-2009). The quantitation of soluble inorganic ions of Ca, Mg, Mn, K, Al and P (defined as the fraction of total ions within cells that is extractable in 5% PCA) was conducted using a simultaneous axial Inductively Coupled Plasma emission spectrophotometer (Vista CCD, Varian, Palo Alto, CA, USA) and Vista Pro software (version 4.0). Supernatants of 3x frozen and thawed samples in PCA were diluted (foliage 100x, sapwood plugs 50x) with distilled deionized water for estimation of soluble ions.

References:

Minocha R, Long S, Magill A, Aber J, McDowell W. Foliar free polyamine and inorganic ion content in relation to soil and soil solution chemistry in two fertilized forest stands at the Harvard Forest, Massachusetts. Plant and Soil. 2000;222:119-37.

Minocha R, Shortle WC, Long SL, Minocha SC. A rapid and reliable procedure for extraction of cellular polyamines and inorganic ions from plant tissues. Journal of Plant Growth Regulation. 1994;13:187-93.

Minocha SC, Robie CA, Khan AJ, Papa NS, Samuelsen AI, Minocha R. Polyamine and ethylene biosynthesis in relation to somatic embryogenesis in carrot (Daucus carota L.) cell cultures. In: Flores HE, Arteca RN, Shannon JC, editors. Polyamines and Ethylene: Biochemistry, Physiology and Interactions: American Society of Plant Physiologists; 1990. p. 339-42.

Minocha R, Long S. Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization. Journal of Chromatography A. 2004;1035:63-73.