Sunlit canopy leaves were collected from four red maple trees in each
of the six CCASE plots using a shotgun on August 3, 2015 and August
14, 2017, during the second and fourth years of treatment in this
study. Three leaves were collected from each tree with petioles
intact, transported on ice and dried to constant mass at 60 °C, before
being homogenized with a mortar and pestle for analysis of nitrogen
and carbon concentrations via flash combustion (Thermoquest NC 2500
autoanalyzer, Thermo Scientific, Waltham, MA, USA).
Immediately after sampling, a pool of ~0.3 g of 6 mm leaf discs was
collected from 2-3 leaves per tree using a paper puncher.
Approximately 0.2 g of foliage were placed in pre-weighed 2 ml
microfuge tubes with 1 ml of cold 5% PCA for extraction of free
polyamines (PAs), amino acids (AAs), and soluble/exchangeable
elements. The remainder of the pooled leaf discs were placed into
separate microfuge tubes to be analyzed for chlorophylls, carotenoids,
soluble protein and sugars. Samples were kept on ice during transport
to the laboratory where they were frozen at -20 °C until further
analyses. Samples in 5% PCA were frozen and thawed three times before
various analyses to disrupt cell membranes and release cell contents
following Minocha et al. (1994).
Free polyamines and amino acids in the 5% PCA extracts were dansylated
and quantified via reverse-phase HPLC per Minocha and Long (2004) with
minor modifications described in Majumdar et al. (2018) .Polyamines
and AAs were analyzed, and the data were processed using PerkinElmer
(Waltham, MA, USA) TotalChrom software (version 6.2.1).
Supernatants of 3x frozen and thawed samples in 5% PCA were diluted
(100x) with distilled deionized water for estimation of
soluble/exchangeable inorganic ions (defined as the fraction of total
ions within cells that is extractable in 5% PCA), including calcium
(Ca), potassium (K), phosphorous (P), magnesium (Mg), manganese (Mn),
aluminum (Al), iron (Fe), and zinc (Zn), Analyses and quantitation of
the elements were conducted using a simultaneous axial Inductively
Coupled Plasma Optical Emission Spectrophotometer (ICP-OES; Vista CCD,
Varian, Palo Alto, CA, USA) and Vista Pro software (version 4.0) per
EPA SW-846 compendium, method 6010.
Using 2 leaf discs, chlorophylls and carotenoids were extracted in 95%
ethanol and detected as per (Minocha et al. 2009). Chlorophyll a,
Chlorophyll b, and total Carotenoids were analyzed with a Hitachi
U2010 spectrophotometer (Hitachi Ltd., Tokyo, Japan; spectral
bandwidth 2 nm, wavelength accuracy of + 0.3 nm, wavelength setting
reproducibility of ± 0.1 nm; with Hitachi UV Solutions software
version 2.0) by scanning absorbance in the range of 350-710 nm.
Equations from Lichtenthaler (1987) were used for quantification.
Soluble protein was extracted from 50 mg of leaf tissue in 500 µl of
Tris extraction buffer by 3 freeze-thaw cycles (Minocha et al. 2019).
The supernatant was quantified for soluble protein concentration per
Bradford (1976) method using Bio-Rad protein assay dye reagent
(Bio-Rad Laboratories, Hercules, CA, USA). Absorbance was recorded at
595 nm with a Hitachi U2010 spectrophotometer (Hitachi Ltd., Tokyo,
Japan; spectral bandwidth 2 nm, wavelength accuracy of +0.3 nm,
wavelength setting reproducibility of ±0.1 nm) and data were analyzed
with Hitachi UV Solutions software version 2.0.
Soluble sugars were extracted from 50 mg of leaf tissue in 80% ethanol
at 65 °C for 30 minutes. Extracts were then filtered through a 0.45 µm
nylon syringe filter. The sugar profiles were determined using
reverse-phase HPLC (Series 200, PerkinElmer, Waltham, MA, USA) coupled
with a refractive index detector (HPLC-RID, Shimadzu Scientific
Instruments Inc., Columbia, MD, USA). 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, USA) was used. Each sugar was quantified using a 5-point external
standard curve (0.125 - 2 mg ml-1). The chromatographs were analyzed,
and the data were processed using PerkinElmer TotalChrom software
(version 6.2.1). To quantify glucose+galactose (two sugars that did
not separate), the areas and concentrations of each were added
together to create a combined standard curve (more detailed methods
are published elsewhere).
References:
Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Analytical Biochemistry 72:248-254
10.1016/0003-2697(76)90527-3I.
Lichtenthaler, H. K. 1987. Chlorophylls and carotenoids: pigments of
photosynthetic biomembranes. Pages 350–382 in L. Packer and R. Douce,
editors. Methods in Enzymology. Academic Press, London.
Majumdar, R., M. Lebar, B. Mack, R. Minocha, S. Minocha, C.
Carter-Wientjes, C. Sickler, K. Rajasekaran, and J. W. Cary. 2018. The
Aspergillus flavus Spermidine Synthase (spds) Gene, Is Required for
Normal Development, Aflatoxin Production, and Pathogenesis During
Infection of Maize Kernels. Frontiers in Plant Science 9
10.3389/fpls.2018.00317I.
Minocha, R. and S. Long. 2004. 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
1035:63-73 10.1016/j.chroma.2004.02.026I.
Minocha, R., S. Long, S. A. Turlapati, and I. Fernandez. 2019. Dynamic
species-specific metabolic changes in the trees exposed to chronic N+S
additions at the Bear Brook Watershed in Maine, USA. Annals of Forest
Science 76:25 10.1007/s13595-019-0808-0I.
Minocha, R., G. Martinez, B. Lyons, and S. Long. 2009. Development of
a standardized methodology for the quantification of total chlorophyll
and carotenoids from foliage of hardwood and conifer tree species.
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