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Sample Collections:

Foliage: foliar samples of red spruce [RS-CY = Current year (first year growth), RS-PY = Past year (second year growth], sugar maple, yellow birch, and American beech were collected from WS1 in locations close to the established lysimeter sites starting in 2000. Samples from the untreated reference site were collected starting in 2004. At both sites, samples were collected from mid to upper canopy by shot gun at three elevational zones designated as: low (500-550 m), mid (550-650 m) and high (650-740 m). Visually healthy needles or leaves were collected for each species (RS-10 trees/year); hardwoods (15 trees (5 trees/elevation/year) at reference site and 20-30 trees at Ca-treated site (5-10 trees/elevation/year). Dominant or co-dominant trees were selected for sampling from a population of 80-90 year-old trees. Needles were chopped into 2-3 mm size pieces using scissors. Disks, 4 mm in diameter were punched with a paper puncher from 2-4 leaves of each hardwood species, avoiding main veins. Two sub-samples were taken from each individual tree’s foliar collection. One sub-sample (approximately 200 mg FW) was placed in a pre-weighed microfuge tube that contained 5% perchloric acid (PCA). The other sub-sample (> 200 mg FW) was kept dry and all samples were transported to the laboratory on ice (Minocha et al. 2000). Samples were stored at -20 ºC until analyzed.

Sapwood plugs:

From 2005-2007 and then again in 2009, sapwood plugs from actively growing sapwood clear (no stained or rotten wood) were extracted from each tree using a drill and an increment hammer. Samples were taken at breast height, avoiding areas with obvious injury to the stem. Three sapwood plugs for broad hardwood trees and 4 for conifer trees were collected from different faces of each tree. A drill was used to first remove the bark and the cambial layer. A dead blow hammer was then used to drive the increment hammer into the exposed sapwood. A plunger was used to extract the sapwood plug into labeled Ziploc bags. Plugs were approximately 1.5 cm in length. Prior to processing, each plug was cut into 4-6 equal segments. After removing any extraneous materials such as bark or discolored wood, samples (~600 mg FW) were transferred into pre-weighed and labeled microfuge tubes. Following the addition of one mL of 5% PCA, samples were stored on ice in the field and frozen at -20oC in the lab until further analysis (Minocha et al. 2015; 2019).

Foliar and sapwood metabolic 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 (2000-2004): 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 (2000-2003): 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 (2004-2015): Both PAs and AAs were simultaneously dansylated and quantified within a single run using the method described in Minocha and Long (2004). 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 (Perkin-Elmer, version 6.2.1).

Exchangeable Inorganic ions (soluble in 5% PCA):

Exchangeable Inorganic ions (2000-2015). 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.

Sapwood plugs:

From 2005-2007 and then again in 2009, sapwood plugs from actively growing sapwood clear (no stained or rotten wood) were extracted from each tree using a drill and an increment hammer. Samples were taken at breast height, avoiding areas with obvious injury to the stem. Three sapwood plugs for broad hardwood trees and 4 for conifer trees were collected from different faces of each tree. A drill was used to first remove the bark and the cambial layer. A dead blow hammer was then used to drive the increment hammer into the exposed sapwood. A plunger was used to extract the sapwood plug into labeled Ziploc bags. Plugs were approximately 1.5 cm in length. Prior to processing, each plug was cut into 4-6 equal segments. After removing any extraneous materials such as bark or discolored wood, samples (~600 mg FW) were transferred into pre-weighed and labeled microfuge tubes. Following the addition of one mL of 5% PCA, samples were stored on ice in the field and frozen at -20oC in the lab until further analysis. Sapwood samples were processed for PA, AA and ion analysis as described above for foliage.

Related Publications:

Schaberg PG, Minocha R, Long S, Halman JM, Hawley GJ, Eagar C (2011) Calcium fertilization at the Hubbard Brook Experimental Forest increases the capacity for stress tolerance and carbon capture in red spruce (Picea rubens) trees during the cold season Trees 25:1053-1061

Minocha R, Long S, Thangavel P, Minocha SC, Eagar C, Driscoll CT (2010) Elevation dependent sensitivity of northern hardwoods to Ca addition at Hubbard Brook Experimental Forest, NH USA Forest Ecology and Management 260:2115-2125

Juice SM et al. (2006) Response of sugar maple to calcium addition to northern hardwood forest Ecology 87:1267-1280