Metabolic labeling of proteins in Arabidopsis was performed according to the protocol reported previously (Skirycz et al., 2011 ). The 1/2 MS media were prepared to contain 14N and 15N isotopes. In the 15N-incorporated medium, the heavy labeling reagents of 15NH415NO3 and K15NO3 (Sigma) were used to replace those containing 14N-isotope. Arabidopsis seedlings were grown at 22°C under long-day conditions (16 : 8 h, light : dark) on 15N- or 14N-isotope-containing medium, respectively. After 2 wk, the seedlings grown on the 14N medium were transferred to a growth chamber at 4°C for 6, 12 and 18 d. As such, 15N-labeled plants at 22°C were used for control experiments (i.e. forward labeling), or the time-course cold-treated plants at 4°C were also metabolically labeled by 15N-isotopes in parallel experiments (i.e. reciprocal labeling) (Kline et al., 2010 ; Qing et al., 2016 (link)). The labeled and unlabeled seedlings were harvested separately, and a mixture with the same amount of 14N- and 15N-isotope-labeled tissues (w/w, 1:1) at the two different temperatures was utilized for relative quantification of proteins.
K15no3
Manufactured by Merck Group
Sourced in United States
K15NO3 is a nitrate compound that can be used as a source of nitrogen in various laboratory applications. It is a white crystalline solid with the chemical formula K15NO3.
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10 protocols using k15no3
1
Isotopic Labeling of Arabidopsis Proteins
2
Anaerobic growth and nitrogen metabolism
3
Sugarcane Denitrification Losses Study
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To test the performance of the novel system under field conditions we investigated the effect of two different fertilizer rates on the magnitude and the partitioning of N2 and N2O denitrification losses from a sugarcane cropping system in subtropical Australia (24°57′53″S, 152°20′0″E). Fertilizer (K15NO3 at 60 atom %, Sigma Aldrich) was applied to the chamber bases at a rate of 50 kg N ha−1 (50 N) and 100 kg N ha−1 (100 N), with four replicates arranged randomly along two sugarcane rows. The fertilizer was dissolved into 1 L of water and applied by hand, to each chamber, to ensure even distribution. Prior to the application of the fertilizer 3 L of water was applied to each chamber to ensure a wet soil profile while minimizing the potential for leaching losses of 15N labelled fertilizer. Following the application of the fertilizer, a 5 cm thick layer of sugarcane trash was placed in each chamber and irrigated with a further 6 L of rainwater. Therefore, a total of 10 L irrigation was added to each chamber, which simulated a 50 mm rain event to completely saturate the soil. The addition of the sugarcane trash is consistent with farming practices in the growing region. More details of the experimental site are given in the Supporting Information.
4
Isotopic Labeling of Extracellular Organic Matter
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13C-labeled and 15N-labeled EOM was generated as described by Stuart et al. (26 (link)), excepting that 1.76 mM K15NO3 (Sigma-Aldrich Co., St. Louis, MO, USA) (98 atom percent 15N) was substituted in ASN, along with 2 mM 13C sodium bicarbonate (Cambridge Isotopes, Tewksbury, MA, USA) (13C; 99%). Bulk isotope ratios for 13C/12C and 15N/14N of a sample of 13C-labeled and 15N-labeled substrate were determined by automated nitrogen and carbon analysis-IRMS (ANCA-IRMS) (PDZE Europa Limited, Crewe, England) at the University of California, Berkeley.
5
Nitrate Uptake Measurement in Seedlings
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15NO3− uptake was measured as described by Wang and Tsay52 (link),88 (link). We cultivated seedlings in six-well plates with 35–40 seeds per well; each plate contained a line. CmCIPK23-OE seedlings were grown in 5 mM KNO3 solution that contained modified MS, 0.5% (w/v) sucrose, and 5 mM KNO3 as the sole N source for 7 days. They were then treated with 10 mM K15NO3 (98% atom 15 N, Sigma, USA) for 30 min and washed in 0.1 mM CaSO4 for 1 min. The seedlings were dried at 80 °C for 2 days. 15N content was analyzed on a stable isotope ratio mass spectrometer (Elementar, ISOprime 100, UK).
6
Measuring 15N Uptake in Wheat Roots
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For 15N uptake analysis, wheat seedlings were cultured in the hydroponic culture (supplemented with 2.5 mM KNO3, pH 5.8) for two weeks. After N starvation by culturing the seedlings in a hydroponic solution without N for 2 days, wheat roots were treated with K15NO3 (98 atom% 15N; SigmaAldrich, number 335134) for 30 min. After washing with 0.1 mM CaSO4 solution and deionized water as described previously38 (link), roots of seedlings were collected and dried at 70 °C for 3 days. After grinding the sample into powder, the 15N content in the root was measured using an isotope ratio mass spectrometer (Thermo Finnigan Delta Plus XP; Flash EA 1112) with three biological replicates.
7
Nitrogen Stable Isotope Labeling in Algae
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D. subspicatus cultures were kept in BBM medium with potassium nitrate as the sole nitrogen source. This salt was supplied in an 15 N-enriched form (98 atom% K15NO3, Sigma-Aldrich). Cell culture conditions, CM preparation, red pigment isolation and detection were the same as described above.
8
Barley Growth and 15N Uptake
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Barley plants were germinated and grown in a matrix composed of vermiculite and sand (4:1, four plants per pot, size 10 ×10 ×11 cm). Seven days after sowing, 50 ml nutrient solutions with 0.3 mM 15NO3− was added for each pot every day. The nutrient solution contained 1 mM KH2PO4, 0.5 mM K15NO3 (60 atom % 15N, from Sigma-Aldrich, Deisenhofen, Germany), 0.5 mM Ca(NO3)2, 0.9 mM MgSO4, 50 µM Fe-EDTA, 16 µM H3BO3, 0.3 µM ZnSO4, 0.3 µM CuSO4, and 0.4 µM Na2MoO4. Leaf samples were taken after 2, 9, and 12 d for 15N measurements.
9
Nitrate Stimulates Spore Germination
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15N-enriched potassium nitrate (0.6 mM; 10 atom% K15NO3, Sigma-Aldrich) was used to examine the role of nitrate in stimulating spore germination. Two milliliters of non-sterile spore suspension with or without 3 ml of E. coli suspension was incubated in 20 ml K15NO3 at 25°C. Resting spores with or without E. coli were cultured in sdH2O without labelling as control. After 0, 3 and 7days of incubation, the resting spores and E. coli were separated by 16% Ficoll 400 (w/v) (Carl Roth GmbH & Co. KG, Germany) at 1,850 rpm for 15 min twice. The separated resting spores were rinsed with sdH2O four times and incubated in the oven at 50°C overnight for isotope analysis. The total N and total 15N abundances of each sample were measured using elemental analyser-isotope-ratio mass spectrometry (EA-IRMS) at the Centre for Stable Isotope Research and Analysis (University of Göttingen, Germany).
10
Arabidopsis Protein Turnover Kinetics
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Seven-day-old Arabidopsis cell culture was transferred from non-labelled media (14N) to media without nitrogen, and the cells washed three times to eliminate 14N in the media. Washed cells were transferred to heavy media (15N) containing two nitrogen sources for optimal growth [16 (link)], namely 1.65 g/l 15NH415NO3 (Sigma–Aldrich 299278) and 1.9 g/l K15NO3 (Sigma–Aldrich 335134). To study protein turnover rate, the labelled Arabidopsis cell culture was collected by vacuum filtration after the first day, third day and fifth day following transfer into new media, then stored at −80°C until use.
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