Organic nitrogen uptake by arbuscular mycorrhizal fungi in a boreal forest.

2.1. Sites Our study site was an upland boreal forest near Delta Junction, Alaska (63°55′ N, 145°44′ W). It had burned in a severe fire in 1999. At the time of sample collection, an AM graminoid ( Festuca altaica Trin.), an AM forb ( Epilobium angustifolium Lam.), ericoid evergreen shrubs ( Ledum palustre L. and Vaccinium vitisidaea L.), and an ericoid deciduous shrub ( Vaccinium uliginosum L.) were common ( Mack et al., 2008 ; Treseder et al., 2004 ). Populus tremuloides Michx. and species of Salix were also abundant; they are deciduous and can each be colonized by AM as well as ectomycorrhizal fungi. Approximately 80% of annual NPP was contributed by plants that form relationships with AM fungi only, and 9% by those that can host both AM and ectomycorrhizal fungi ( Mack et al., 2008 ). Permafrost is discontinuous in this area and was not present in the site. The local climate was cold and dry, with a mean annual temperature of −2 °C and a precipitation rate of 303 mm y −1 ( http://weather.noaa.gov/ ). The site consisted of a block design described in Treseder et al. (2007) . Each block contained an N-fertilized plot and a control plot. The N-fertilized plots were originally dosed with 200 kg N ha −1 y −1 in June 2002, and thereafter received 100 kg N ha −1 y −1 in June of each year. We sampled in three of these blocks ( n = 3), chosen at random. Our replicate number was limited owing to the immediate availability of QDs at the field site. 2.2.

Preparation of quantum dots

To conjugate QDs to organic nitrogen substrates, we used the same methods and concentrations as described by Whiteside et al. (2009) . Briefly, commercial green (535 nm emission) and orange (620 nm emission) carboxyl QDs were purchased from Vive Nano (Ontario, Canada). Green QDs were bound to the amino groups of glycine (MP Biomedical, Solon, Ohio, USA), and orange QDs were bound to the amino groups of chitosan ( Fig. 1 ; MP Biomedical). Quantum dot size (i.e., the average hydrodynamic diameter) was estimated using RICS and the Stokes–Einstein equation ( Digman et al., 2005 ; Uhlenbeck and Ornstein, 1930 ). Diameters of the green QDs averaged ~3 nm; orange QDs ~8 nm. Approximately five glycine molecules were conjugated to each green QD, and one chitosan molecule to each orange QD. Amino terminated QDs were prepared in the same manner, except using low molecular weight poly(allylamine hydrochloride) in place of glycine or chitosan ( Jin and Gao, 2009 ). These QDs were solely used to represent mineralized forms of QD-glycine and QD-chitosan during spectral analysis (see below). They were not included as field injections. 2.3. Field incubations To assess uptake of organic nitrogen by AM fungi in the field, on September 9, 2008 we injected two QD cocktails into each of the N fertilized and control plots ( Table S1 ). The first cocktail contained an equal (0.1 μM) mixture of green-labeled glycine and orange-labeled chitosan. The second cocktail contained an equal mixture of orange and green QD controls, which consisted of unbound QDs subjected to the same conditions as the labeled conjugates, but lacking the binding reagent. Twenty ml of each (0.1 μM) QD cocktail were injected ~1 cm into the soil. Each plot received either two injections of QD controls plus two injections of glycine-bound QDs mixed with chitosan-bound QDs, or three injections of each. Twenty-four hours following injection, 2 cm diameter × 10 cm deep soil cores were taken from each injection point. In addition, in each plot, one “uninjected” core was collected from a random area of no injection, for a total of 34 cores. QD-glycine and QD-chitosan were stored separately and mixed immediately before injection. The soil corer was cleaned with sodium hypochlorite between samples. Cores were initially frozen at −4 °C and stored at −80 °C. 2.4. Arbuscular mycorrhizal colonization of roots We used wet sieving through 1 mm mesh to extract all fine (