uid=HFR,o=lter,dc=ecoinformatics,dc=org all public read doi:10.6073/pasta/db695dd1bcd61ccc45fcf3a816b24ce3 Andrew Richardson https://orcid.org/0000-0002-0148-6714 2023 English

We know surprisingly little about whole-tree nonstructural carbon (NSC; primarily sugars and starch) budgets. Even less well understood is the mixing between recent photosynthetic assimilates (new NSC) and previously stored reserves. And, NSC turnover times are poorly constrained. We characterized the distribution of NSC in the stemwood, branches, and roots of two temperate trees, and we used the continuous label offered by the radiocarbon (14C) bomb spike to estimate the mean age of NSC in different tissues. NSC in branches and outermost stemwood growth rings had the 14C signature of the current growing season. However, NSC in older above- and below-ground tissues was enriched in 14C, indicating that it was produced from older assimilates. Radial patterns of 14C in stemwood NSC showed strong mixing of NSC across the youngest growth rings, with limited.

carbohydrates carbon tree physiology LTER controlled vocabulary primary production LTER core area Harvard Forest HFR LTER USA HFR default

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Creative Commons Zero v1.0 Universal https://spdx.org/licenses/CC0-1.0.html CC0-1.0 https://harvardforest.fas.harvard.edu/exist/apps/datasets/showData.html?id=hf226 Tom Swamp Tract (Harvard Forest). Coordinates based on WGS84 datum. -72.218 -72.218 +42.513 +42.513 250 250 meter 2012 2012 genus Pinus species strobus white pine genus Quercus species rubra red oak complete Information Manager Harvard Forest

324 North Main Street Petersham MA 01366 USA

(978) 724-3302 hf-im@lists.fas.harvard.edu Harvard Forest

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We selected one tree of each species for destructive harvesting. The main criteria for selection were (1) evidence of vigorous growth; and (2) a well-developed crown that was not overtopped by surrounding trees. The pine was 12 m high, 23 years of age, 18.5 cm diameter at breast height (DBH), and established following the harvest in 1990. The oak was 20 m high, 30 years of age, and 17.0 cm DBH, and established following the thinning in 1983. Trees were felled and stemwood bolts (ca. 50 cm in length) collected at breast height, mid-way up the stem, and near the top of the stem. We also collected current-year twigs, as well as multi-year branches, from throughout the crown. We then partially excavated the stump and root system of each tree, and collected samples of large coarse roots (diameter greater than 5 cm), as well as medium coarse roots (approx. 1 cm) and fine roots (no more than 1 mm) that were directly attached to the large coarse roots (and thus undoubtedly from the same tree). Samples were processed in the laboratory on the same day that they were collected in the field. Cookies, or stemwood sections approximately 2 cm in thickness, were cut from the stemwood bolts. Using a hammer and chisel, we separated the outer bark and phloem, and then separated individual annual growth rings from each cookie. We identified the heartwood-sapwood transition based on wood color. In the large and intermediate coarse roots we could not easily count rings. We therefore separated the large coarse root wood by thirds into three depths, with D1 denoting the outer (most recent) wood and D3 the center (oldest) wood. For all root samples, in lieu of ring counts we determined the mean age of the cellulose (i.e. structural C) via 14C analysis as described below. Samples were frozen at -80 deg C and then freeze-dried (FreeZone 2.5, Labconco, Kansas City, MO USA, and Hybrid Vacuum Pump, Vacuubrand, Wertheim, Germany) before being homogenized and milled to 20 mesh (Wiley Mini Mill, Thomas Scientific, Swedesboro, NJ USA). Samples were then kept at -80 deg C until the NSC extraction was conducted. Additional sample material was kept frozen at -80 deg C.

For total soluble sugar concentration (following Chow and Landhausser, 2004), 50 mg of tissue was freeze-dried a second time (24 hours; for determination of precise weight) and subjected to hot ethanol extraction followed by colorimetric analysis with phenol-sulfuric acid. The resulting extract was read at 490 nm with a microplate reader (Epoch Microplate Spectrophotometer, Bio-Tek Instruments, Winooski, VT, USA) with sugar concentration (expressed as mg sugar per g dry wood) calculated from a standard curve of 1:1:1 glucose-fructose-galactose (Sigma Chemicals, St. Louis, MO, USA). For starch analysis (following Wargo et al., 2002), the remaining tissue residue was boiled in KOH followed by neutralization with acetic acid and enzymatic digestion with amyloglucosidase. Glucose hydrolyzate was determined using a glucose hexokinase kit (Pointe Scientific, Canton, MI, USA) and read at 340 nm with the microplate reader. Starch concentration (expressed as mg starch per g dry wood) was calculated based on glucose standard (Pointe Scientific, Canton, MI, USA) curves.

Following Richardson et al. (2013) and Carbone et al. (2013) we used the 14C bomb spike to estimate the age of extracted NSC, and in the case of root wood, which could not be easily aged by ring counting, to estimate the age of the cellulose (i.e. structural C). The bomb spike method is based on the fact that above-ground testing of thermonuclear weapons between 1955 and 1963 approximately doubled the amount of 14CO2 in the atmosphere (Levin and Kromer, 2004). Since 1963, when the Limited Nuclear Test Ban Treaty was signed, the 14C content of atmospheric CO2 has decreased through mixing with oceanic and terrestrial C reservoirs, and by addition of 14C-free CO2 from fossil fuel burning (Levin et al., 2010). The C in photosynthetic products reflects the 14C content of atmospheric CO2 in the year assimilation occurred, and hence these products are labeled with a unique isotopic signature. Thus, the mean 14C age of a sample can be determined by measuring its 14C content, and comparing this to the atmospheric 14CO2 record. We conducted 14C analysis on sugars and starch extracted following Czimczik et al. (2014). Soluble NSC (mostly sugars) was isolated by hot water extraction, and insoluble NSC (mostly starch) was isolated by acid digestion following lipid removal by boiling in ethanol. The extractions were conducted sequentially from each tissue sample. We conducted the 14C analysis on cellulose extracted using a Soxhlet apparatus (Leavitt and Danzer, 1993). For the 14C analysis of both NSC and cellulose, extracts were combusted to CO2, purified on a vacuum line, and converted to graphite (Xu et al., 2007). Graphite was analyzed for its 14C content at the W.M. Keck Carbon Cycle Accelerator Mass Spectrometry facility at UC Irvine (Southon et al., 2007).

Table 1. Concentrations (mg per g of oven dry wood) of sugar and starch in woody tissues of white pine and red oak. Values are mean and 1 SD, calculated across the three plate wells read for each sample. Table 2. Radiocarbon age of extracted sugar, starch, and cellulose, from tissues of white pine and red oak. Delta 14C was measured at the W.M. Keck Carbon Cycle Accelerator Mass Spectrometry facility at UC Irvine. Reported uncertainties are 1 SD of the analytical uncertainty. 14C data were converted to age estimates using the bomb spike method. Asterisks denote samples for which multiple extractions were run independently.

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(978) 724-3302 (978) 724-3595 https://harvardforest.fas.harvard.edu https://ror.org/059cpzx98 pointOfContact The Harvard Forest Long-Term Ecological Research (LTER) program examines ecological dynamics in the New England region resulting from natural disturbances, environmental change, and human impacts. National Science Foundation LTER grants: DEB-8811764, DEB-9411975, DEB-0080592, DEB-0620443, DEB-1237491, DEB-1832210. hf226-01-concentrations.csv concentrations hf226-01-concentrations.csv 4671 60140ee3a23fd265837250f6bcf350fe 1 \r\n column , https://harvardforest.fas.harvard.edu/data/p22/hf226/hf226-01-concentrations.csv species species name species name NA missing value tissue.sample tissue sample tissue sample NA missing value sugar.mean mean concentrations (mg per g of oven dry wood) of sugar in woody tissues dimensionless 0.1 real NA missing value sugar.sd standard deviation of concentrations (mg per g of oven dry wood) of sugar in woody tissues dimensionless 0.1 real NA missing value starch.mean mean concentrations (mg per g of oven dry wood) of starch in woody tissues dimensionless 0.1 real NA missing value starch.sd standard deviation of concentrations (mg per g of oven dry wood) of starch in woody tissues dimensionless 0.1 real NA missing value 82 hf226-02-radiocarbon-age.csv radiocarbon age hf226-02-radiocarbon-age.csv 4607 c24af2d7fd681cede93bd054918f25b8 1 \r\n column , https://harvardforest.fas.harvard.edu/data/p22/hf226/hf226-02-radiocarbon-age.csv species species species NA missing value extract extract extract NA missing value tissue tissue sample tissue sample NA missing value a14c radiocarbon age dimensionless 0.1 real NA missing value d14c.sd standard deviation of uncertainty of radiocarbon age dimensionless 0.1 real NA missing value e14c.age radiocarbon age estimates nominalYear 0.1 real NA missing value 80 community short-term measurement https://harvardforest.fas.harvard.edu/exist/apps/datasets/showData.html?id=hf176