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Quantifying remobilization of pre-existing nitrogen from cuttings to new growth of woody plants using… Kalcsits, Lee A; Guy, Robert D Jul 12, 2013

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METHODOLOGY Open AccessQuantifying remobilization of pre-existingnitrogen from cuttings to new growth of woodyplants using 15N at natural abundanceLee A Kalcsits and Robert D Guy*AbstractBackground: For measurements of nitrogen isotope composition at natural abundance, carry-over of pre-existingnitrogen remobilized to new plant growth can cause deviation of measured isotope composition (δ15N) from theδ15Nof newly acquired nitrogen. To account for this problem, a two-step approach was proposed to quantify andcorrect for remobilized nitrogen from vegetative cuttings of Populus balsamifera L. grown with either nitrate(δ15N = 58.5‰) or ammonium (δ15N = −0.96‰). First, the fraction of carry-over nitrogen remaining in the cuttingwas estimated by isotope mass balance. Then measured δ15N values were adjusted for the fraction of pre-existingnitrogen remobilized to the plant.Results: Mean plant δ15N prior to correction was 49‰ and −5.8‰ under nitrate and ammonium, respectively.Plant δ15N was non-linearly correlated to biomass (r2 = 0.331 and 0.249 for nitrate and ammonium, respectively;P < 0.05) where the δ15N of plants with low biomass approached the δ15N of the pre-existing nitrogen.Approximately 50% of cutting nitrogen was not remobilized, irrespective of size. The proportion of carry-overnitrogen in new growth was not different between sources but ranged from less than 1% to 21% and wasdependent on plant biomass and, to a lesser degree, the size of the cutting. The δ15N of newly acquired nitrogenaveraged 52.7‰ and −6.4‰ for nitrate and ammonium-grown plants, respectively; both lower than their sourcevalues, as expected. Since there was a greater difference in δ15N between the carried-over pre-existing and newlyassimilated nitrogen where nitrate was the source, the difference between measured δ15N and adjusted δ15N wasalso greater. There was no significant relationship between biomass and plant δ15N with either ammonium ornitrate after adjusting for carry-over nitrogen.Conclusion: Here, we provide evidence of remobilized pre-existing nitrogen influencing δ15N of new growth ofP. balsamifera L. A simple, though approximate, correction is proposed that can account for the remobilized fractionin the plant. With careful sampling to quantify pre-existing nitrogen, this method can more accurately determinechanges in nitrogen isotope discrimination in plants.Keywords: Nitrogen remobilization, Poplar, δ15NBackgroundMeasurements of nitrogen isotope composition at nat-ural abundance (δ15N-the 15N:14N ratio of a sample rela-tive to the isotope ratio of a known internationalstandard (air N2)), expressed in per mil, may be affectedby remobilization of previously acquired nitrogen intonew plant growth. To determine the δ15N of newlyacquired nitrogen, the carry-over of pre-existing nitro-gen must be considered. The use of δ15N at naturalabundance has increased in plant physiology andecology where small but tractable changes δ15N ofplants or plant tissues can indicate changes in soil ni-trogen sources or changes in nitrogen-use physiology[1-6]. Quantifying temporal changes in plant nitrogensources in the field or plant nitrogen isotope fractionationrequires confidence in the measurement of the δ15N ofnewly acquired nitrogen. When not working at the naturalabundance level, as in many 15N enrichment experiments,* Correspondence: rob.guy@ubc.caDepartment of Forest Sciences, University of British Columbia, 2424 MainMall, Vancouver, BC V6T1Z4, CanadaPLANT METHODS© 2013 Kalcsits and Guy; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.Kalcsits and Guy Plant Methods 2013, 9:27http://www.plantmethods.com/content/9/1/27knowledge of the precise isotopic composition of pre-existing nitrogen may not be necessary because newlyacquired nitrogen will have a markedly different iso-topic signature [3,7].Carried-over pre-existing nitrogen in a plant can beremobilized to growing tissue. For example, during estab-lishment of sexually and vegetatively propagated plants,stored nitrogen is remobilized from the propagule (seedor cutting) to the newly growing tissue as a nitrogen sup-ply until roots are capable of supporting the nitrogendemands of the plant [7]. In woody perennials, nitrogenfrom senescing leaves accumulates in stem tissue duringautumn to be re-used by developing tissues during re-growth in the spring [8,9]. Nitrogen transported to newshoot and/or root growth come from vegetative storageproteins kept over the dormant period [10]. Vegetativestorage proteins can account for a sizeable fraction of totalnitrogen in dormant stem tissue [11,12]. These vegetativestorage proteins, in addition to other mobile nitrogen frac-tions in stem tissue can be remobilized to shoots or rootsof plants. If the δ15N of remobilized nitrogen is differentfrom the δ15N of newly acquired nitrogen, it may influ-ence the measured δ15N of new growth.Although the δ15N of carried over nitrogen may pro-vide an isotopic signature to compare against newgrowth δ15N, within a bulk nitrogen pool (e.g., a cut-ting), there may be differences in δ15N between variousamino acids and other nitrogen-containing compounds[13-15]. This may impact the δ15N of remobilized nitro-gen relative to the δ15N of the bulk cutting prior to newgrowth if the δ15N of the remobilized fraction is differ-ent from the non-remobilized fraction of the cutting.However, if much of the carried-over pre-existing nitro-gen contributes to the growth of new plant tissue, theδ15N of the remobilized nitrogen should resemble theδ15N of the bulk source tissue because many, and cer-tainly the most predominant, nitrogen-containing com-pounds (e.g. vegetative storage proteins) will contributeto remobilized nitrogen from the source tissue (cutting)to new growth. If there was any isotope discriminationassociated with remobilization itself, as consumption ofa nitrogen pool approaches completion, the transferredfraction (the product) would then approach the δ15N ofthe stored fraction (the source).Here, a two-step mass balance approach is proposedto account for carry-over nitrogen remobilized from pre-existing nitrogen pools to growing tissues in plants usingPopulus balsamifera L. (balsam poplar) grown with ei-ther ammonium or nitrate. The two nitrogen sourcesbracketed the δ15N of pre-existing N, with the nitratebeing enriched in 15 N relative to the cutting and theammonium being depleted relative to the cutting. There-fore, if carried-over nitrogen from pre-existing nitrogenhas an appreciable effect on newly assimilated nitrogenisotope values, opposing relationships with biomassshould be evident. Poplars in general have long beenused as model systems in tree biology and genetics, andPopulus trichocarpa Torr. & Gray, (syn: P. balsamiferassp. trichocarpa) was the first woody species to have itsgenome completely sequenced [16]. The approach de-scribed here is applicable not just to plants grown fromcuttings, but also, with little adjustment, to plants grownfrom seed.Resultsδ15N, growth and nitrogen concentrationBoth ammonium and nitrate-grown plants were depletedin 15 N relative to the source (Table 1). Under nitrate,mean root, stem and leaf δ15N was 44.4, 47.1 and 50.1‰whereas for ammonium, these values were −8.6, -6.6 and−5.1‰, respectively. The vegetative cuttings used topropagate the plants were not different between sourcesand had a mean δ15N of 0.92‰ at the start of the experi-ment. During plant growth, the δ15N of the cuttingsmoved towards and/or past the isotope signature of thesource (Table 1). For nitrate-grown plants, cuttingsbecame enriched (23.5‰) and for ammonium-grownplants, cuttings became depleted (−3.1‰) relative totheir starting isotopic composition (P < 0.05). Genotypicmeans for whole plant biomass ranged from 0.51 to4.46 g and 0.33 to 3.48 g under nitrate and ammonium,respectively (Table 2). There was a significant correlationbetween genotype means for biomass on nitrate versusammonium (r = 0.54, P < 0.05) whereby clones that grewwell on one source also grew well on the other. Therange in mean genotype biomass provided a range ofisotopic dilutions to determine the influence of carried-over pre-existing nitrogen on whole-plant and organTable 1 Mean measured and, for new tissues, adjustedroot, stem, leaf and cutting δ15N values (‰ ± SE; N = 75)of Populus balsamifera L. hydroponically grown withnitrate (58.5‰) or ammonium (−0.96‰)Nitrate AmmoniumMeasured δ15NPlant 49.04 ± 0.43 −5.81 ± 0.16Root 44.42 ± 0.42 −8.59 ± 0.15Stem 47.08 ± 0.40 −6.62 ± 0.18Leaf 50.10 ± 0.44 −5.07 ± 0.15Cuttingstart 0.87 ± 0.35 0.97 ± 0.31Cuttingend 23.47 ± 0.88 −3.10 ± 0.23Adjusted δ15NPlant 52.68 ± 0.26 −6.43 ± 0.19Root 47.87 ± 0.28 −9.45 ± 0.19Stem 50.76 ± 0.26 −7.29 ± 0.19Leaf 54.01 ± 0.25 −5.59 ± 0.18Kalcsits and Guy Plant Methods 2013, 9:27 Page 2 of 9http://www.plantmethods.com/content/9/1/27level δ15N. When the unadjusted δ15N for new growthof the whole plant was plotted against biomass, therewas a significant non-linear relationship (r2 = 0.331 and0.249 for nitrate and ammonium, respectively; P < 0.05)(Figure 1). The unadjusted δ15N for new growth of thewhole plant for genotypes with low biomass were closerto the δ15N of cutting nitrogen. In contrast, the δ15N ofgenotypes with high biomass were closer to the δ15N ofnewly acquired nitrogen, which resembles the source butdoes not equal it due to isotope discrimination that occursduring uptake and assimilation. Nitrogen concentrationwas not different between cuttings before the start of theexperiment, but was significantly greater at the end of theexperiment when grown with ammonium as compared tonitrate (Table 3). Root nitrogen concentration was not sig-nificantly different between sources but stem and leafnitrogen concentrations were greater when grown withammonium (P < 0.05).Carry-over nitrogen in new plant growth is dependent onthe size of the nitrogen pool in the cutting relative to thenitrogen pool in the new growthThe δ15N of vegetative cuttings at the end of the experi-ment was approximately halfway between the startingcutting δ15N and the δ15N of newly formed organs, indi-cating that some considerable portion of the originalnitrogen was retained within the cuttings. From equa-tion 4, the non-remobilized fraction of the nitrogencontained within the cuttings at the end of the ex-periment averaged 50% and 52% for nitrate and ammo-nium, respectively and was not significantly different.Therefore, approximately half of the starting nitrogen wasremobilized for new growth (Figure 2). The mean quantityof nitrogen remobilized was 3.2 mg but was dependent onthe size of the cutting (r = 0.057; P < 0.05; data not shown).There was no source effect on the proportion of carry-over, which averaged 8% but decreased non-linearly from21% to 1% with the biomass of new growth (not shown).The difference between source δ15N and the cutting δ15Nat harvest was greater for nitrate than ammonium(Table 1), causing carry-over nitrogen to have a greater in-fluence on measured plant δ15N under nitrate (Figure 3A)than ammonium (Figure 3B).Relationships between plant δ15Nand biomass were notsignificant after accounting for carry-over of pre-existingnitrogenThere were significant linear relationships between thecontribution of nitrogen carried-over from the cuttingTable 2 Mean root, stem, leaf, cutting and whole plantbiomass (g ± SE; N = 75) of Populus balsamifera L.hydroponically grown with nitrate or ammoniumBiomass (g)Nitrate AmmoniumRoot 0.18 ± 0.02 0.20 ± 0.01Stem 0.40 ± 0.03 0.29 ± 0.02Leaf 1.28 ± 0.09 1.15 ± 0.07Cuttingstart 0.49 ± 0.03 0.54 ± 0.04Cuttingend 0.67 ± 0.06 0.65 ± 0.05Whole Plant 1.86 ± 0.14 1.64 ± 0.10Figure 1 Unadjusted (open symbols) and adjusted (closedsymbols) δ15N for new growth of the whole plant plottedagainst biomass for Populus balsamifera L. grown with eithernitrate (A) or ammonium (B). Each data point represents agenotypic mean (N = 3). Correlation coefficients and P-values areplaced near each line.Table 3 Mean nitrogen concentrations (μmol g dw-1 ± SE;N = 75) of roots, stems, leaves and cuttings of Populusbalsamifera L. hydroponically grown with nitrate orammoniumTissue nitrogen concentration (μmol g dw-1)Nitrate AmmoniumRoot 2.50 ± 0.04 2.55 ± 0.03Stem 0.41 ± 0.01 0.76 ± 0.03Leaf 1.84 ± 0.03 2.19 ± 0.04Cuttingstart 0.83 ± 0.01 0.83 ± 0.02Cuttingend 0.59 ± 0.03 0.71 ± 0.02Kalcsits and Guy Plant Methods 2013, 9:27 Page 3 of 9http://www.plantmethods.com/content/9/1/27and the unadjustedδ15N for new growth of the wholeplant (P < 0.05) (Figure 3). Relative to the measuredδ15N of each corresponding organ, newly assimilatednitrogen was more depleted in 15 N under ammoniumand more 15 N enriched under nitrate. After accountingfor the presence of remobilized nitrogen, the adjustedroot, stem and leaf δ15N values were 47.87, 50.76 and54.01‰ and −9.45, -7.29 and −5.59‰ for nitrate andammonium, respectively. This represented an approxi-mate shift of 3.5 to 4‰ and 0.5 to 0.8‰ for nitrate andammonium grown plants, respectively. There was no re-sidual relationship between biomass and newly ac-quired plant δ15N (i.e., non-significant regressions inFigure 3) indicating that the carry-over nitrogen waswell accounted for.The δ15N of remobilized nitrogen to new growth is notsignificantly different than the δ15Nof the cuttingWhen cuttings of randomly selected Populus trichocarpaTorr. & Gray genotypes were flushed without exogenoussources of nitrogen, the nitrogen isotope composition ofremobilized nitrogen was not significantly different thanthe isotope composition of complementary cuttingssampled prior to bud flush (Table 4). The isotope com-position of the bud was significantly greater (P < 0.05)than the cutting stem (2.44 vs 2.11‰) and accounted forapproximately 10% of the nitrogen in the vegetative cut-tings. After flushing, the isotope composition of the shootwas not significantly different than the cutting stem (P >0.05) and similar to our estimates using isotope mass bal-ance, approximately 50% of the nitrogen from the cuttingstem and bud was allocated to the shoot (data not shown).Since a greater portion of nitrogen was allocated to theshoot after flushing, there was a corresponding 40% de-crease in the nitrogen content of the cutting stem afterflushing. Although there was significant genotypic vari-ation in the isotope composition of the cuttings, there wasno significant difference in the amount of nitrogenremobilized from the cuttings (P > 0.05).DiscussionThe objective was to quantify and correct for carry-overnitrogen from cuttings in new growth of P. balsamiferahydroponically grown under steady-state conditions.Here, it was shown that nitrogen remobilized from cut-tings influences plant δ15N. Carry-over nitrogen wouldbe inconsequential for plants grown from relatively smallseeds or in longer-term experiments, where the totalaccumulated nitrogen pool represents almost all of theFigure 2 Per cent of pre-existing nitrogen remobilized into newgrowth (A) and per cent contribution of carried-over pre-existing cutting nitrogen to total plant nitrogen (B) in Populusbalsamifera L. grown with either nitrate or ammonium. Bars aremeans ± SE (N = 75).Figure 3 Unadjusted (open symbols) and adjusted (closedsymbols) δ15N for new growth of the whole plant plottedagainst the per cent contribution of carry-over nitrogen to newgrowth of Populus balsamifera L. supplied with either nitrate(A) or ammonium (B). Each data point represents a genotypicmean (N = 3). Correlation coefficients and P-values are placed neareach line.Kalcsits and Guy Plant Methods 2013, 9:27 Page 4 of 9http://www.plantmethods.com/content/9/1/27nitrogen within the plant. However, in cases where bio-mass accumulation is low relative to the size of thepropagule, without correction, deviation of measuredδ15N from the δ15N of newly acquired nitrogen can leadto misinterpretations in physiological and ecologicalstudies. It has been suggested that there are carry-overeffects contributing to interannual variability in carbonisotope signatures (δ13C) in woody plants [17,18] but asfar as we know, aside from [19], this is the first attemptto quantify the carry-over effect on nitrogen isotopecomposition of new growth in woody plants.Accounting for remobilized nitrogen from the cuttingto the root, stems and leaves required a two-step ap-proach. Since vegetative cuttings are a part of the stem,nitrogen contained within the cutting should be com-bined with the general stem nitrogen pool. However,there is a certain fraction of nitrogen in the vegetativecutting that was not mobilized and therefore, needs tobe estimated to calculate the corresponding amount ofcutting nitrogen that was remobilized into the newgrowth. In the present study, and noting that someisotope discrimination is expected during nitrogen up-take and assimilation [4,6,14,20], the presence of non-remobilized nitrogen in the cutting was indicated bya difference between stem and cutting δ15N that wasweighted towards the source δ15N (Table 1). Previouswork has indicated that there is often a fraction that is notremobilized to sink tissue [8]. Once non-remobilizednitrogen and the proportion of cutting nitrogen that ispart of the stem nitrogen pool had been partitioned, theamount of nitrogen mobilized into the plant nitrogen poolwas quantified. To make this partitioning calculation, itwas assumed that new nitrogen in the cutting was mixedwith remobilized (carried-over) nitrogen from the cuttingsimilar to that of stem tissue. Since there was influx ofnewly assimilated nitrogen into the vegetative cutting, asindicated by a growth dependent change of cutting δ15Ntowards the source δ15N, it would be reasonable toassume there is a certain degree of mixing of nitrogenwithin the mobile stem nitrogen pool.Variation in the difference in δ15N between measuredand newly acquired nitrogen may, in part, be a conse-quence of assuming that the distribution of carry-overnitrogen was proportionate to the nitrogen allocationbetween roots, stems and leaves. Dong S, 2004 [7]reported that approximately 30% of remobilized nitrogenwas allocated to the root and 70% was allocated to theshoot in vegetatively propagated poplar, which favorsshoots over roots slightly less than our results. Althoughtotal nitrogen contents and biomass of roots and shootswere not reported in [7], if the root:shoot ratios weresimilar to our results, then this would be consistent witha proportional allocation of remobilized nitrogen tothose organs. By assuming proportionate allocation ofcarry-over nitrogen, we acknowledge that there may besome error associated with this assumption but the over-all effect of the associated error is small. In situationswhere carry-over nitrogen accounted for a large por-tion of plant nitrogen, unequal allocation could havean appreciable effect. However, in our case where, inmost cases, carry-over nitrogen accounted for lessthan 15% of plant nitrogen, the influence on adjustedisotopic composition for roots, stems and leaveswould be mostly inconsequential.Another assumption made was to equate the δ15N ofremobilized nitrogen with the δ15N of the cutting at thestart of experiment. To validate this assumption, vegeta-tive cuttings from four randomly chosen genotypes froma common garden experiment for P. trichocarpa weregrown without exogenous nitrogen for 21 days. Theestimates of the amount of nitrogen remobilized fromthe cutting to the new growth were similar to thoseobtained using our proposed isotopic mixing model andcorrection. Using a different approach, [19] reportedthat there was no difference in remobilized δ15N andstored δ15N, and that no fractionation occurred duringremobilization. Although biochemical variation in δ15Noccurs in plants [13-15] and these variations may contrib-ute to error to our approach, our results indicate that itwould seem to be small relative to the correction applied.The proportion of soluble nitrogen in cuttings maylimit nitrogen remobilization which may provide anexplanation for why approximately half of pre-existingnitrogen was not remobilized. During the dormantperiod, nitrogen storage proteins accumulate in stem tis-sue and the proportion of soluble, stored nitrogenTable 4 Nitrogen isotope composition of vegetative cuttings, pre-flush and post-flush (‰ ± SE; N = 5 except for roots(N = 2-5) where replicates were combined when root biomass was too low for analysis), for four randomly selectedPopulus trichocarpa Torr. & Gray genotypes flushed in ddH2O containing no exogenous sources of nitrogen for 21 daysPre-flush cutting Post-flush cuttingGenotype CuttingStem δ15N Bud δ15N CuttingStem δ15N Shoot δ15N Root δ15NLILD 26-3 2.34 ± 0.17 1.97 ± 0.27 2.11 ± 0.34 2.53 ± 0.13 3.08 ± 0.02LILD 26-5 1.68 ± 0.16 1.97 ± 0.24 1.63 ± 0.36 1.63 ± 0.06 2.46 ± 0.04TOBA 23-3 3.35 ± 0.26 2.25 ± 0.18 2.82 ± 0.46 2.88 ± 0.08 3.02 ± 0.06PHLA 22-1 2.35 ± 0.17 2.28 ± 0.16 2.34 ± 0.37 2.41 ± 0.08 2.20 ± 0.26Kalcsits and Guy Plant Methods 2013, 9:27 Page 5 of 9http://www.plantmethods.com/content/9/1/27increases [21-24]. Estimates of soluble nitrogen in stemsof Fraxinus excelsior were between 60 and 70% during thedormant period [20]. Our natural abundance nitrogen iso-tope mass balance approach indicates that approximately50% of total cutting nitrogen was remobilized to sink tis-sues (leaves, roots and stems). If the proportion of solublenitrogen in P. balsamifera is similar, then only a smallfraction of soluble nitrogen was not remobilized to newgrowth. For hybrid poplar, between 60 and 70% of nitro-gen in cuttings was remobilized into actively growing tis-sue [7]. This proportion was comparable with estimates ofremobilization of nitrogen from source tissue (leaves) ofBrassica napus to sinks (developing siliques) [25].The use of two separate nitrogen sources with δ15Nvalues both greater and lower than the δ15N of thecutting at the start of the experiment provided somevalidation for the correction. Previously, enriched nitrogenapplied as 15 N allowed for the quantification of remo-bilized nitrogen into growing plant tissue [7]. In thepresent study, newly acquired nitrogen was approximately5 to 10‰ depleted relative to the cutting when plantswere grown on ammonium (δ15N = −0.96 ‰), and 50to 55‰ enriched when plants were grown on nitrate(δ15N = 58.5 ‰). This contrast in the isotopic com-position of two sources of newly acquired nitrogen,relative to nitrogen that previously existed in the cut-ting, produced negative and positive non-linear rela-tionships between biomass and the unadjusted plantδ15N. Here, after correction, biomass had no impacton adjusted plant δ15N for either source and any ob-served relationship was primarily a result of nitrogencarried-over from the cutting. Our approach workedwell either way, which should not be the case if theδ15N of remobilized nitrogen were consistently offsetin one direction from the δ15N of bulk nitrogen.ConclusionHere, we provided evidence that remobilized nitrogencarried-over from stem cuttings can affect measuredδ15N of new growth in poplar, a perennial model plant.Carry-over nitrogen from pre-existing nitrogen poolsneeds to be considered when measuring δ15N at naturalabundance. By applying a two-step mass balance correc-tion, the contributions of carried-over pre-existing nitro-gen to the nitrogen content and δ15N of new growth canbe quantified and accounted for. This, in turn, will allowfor a better assessment of isotope discrimination associ-ated with the acquisition of new nitrogen from therooting environment and could be helpful in physiologyand ecology studies measuring nitrogen isotope compos-ition at natural abundance to address questions relatingto nitrogen use dynamics at the soil or plant level. Themethods presented here are not only applicable to thisparticular species or set of conditions but, with carefulsampling protocols, can be applied to other experimentalsystems that require the measurement of δ15N at naturalabundance during plant growth and development.MethodsPlant material and experimental designFirst year branches of 25 genotypes of Populus balsamiferaL. ranging from 51°N to 56°N from the AgricultureCanada Balsam Poplar (AgCanBaP) collection [26] wereobtained from the AAFC-AESB Agroforestry DevelopmentCentre at Indian Head, Saskatchewan, Canada andstored at 4°C for approximately three months to fulfillchilling requirements. The five provenances reflecteda climatic gradient for the species that extends froma prairie ecosystem to the boreal forest of the CanadianShield; namely Outlook (OUT; 51.1°N, 106.2°W), Sas-katoon (SKN; 2.2°N, 106.4 °W), Turtleford (TUR;53.2°N, 108.3 °W), Cold Lake (CLK; 54.2°N, 110.1°W)and Gillam (GIL; 56.4°N, 94.7 °W). Two-node cut-tings, approximately 6–8 cm long were weighed forfresh weight and arranged in a randomized completeblock design with three blocks of two nitrogen treatmentssupplied as either 250 μM Ca(NO3)2 or 250 μM(NH4)2SO4. Plants were grown for 45 days in ahydroponics solution until harvest. Complementarysamples of each genotype were collected (N = 3) todetermine initial isotope composition and nitrogenconcentration.Hydroponics systemThe hydroponics system was comprised of six 1000 Lcontainers lined with rubber pond liner material(Firestone, Nashville, TN, USA) set up in a green-house under ambient light conditions supplementedby sodium halide lighting (600 μmol m-2 s-1 PPFD)and 18/6 photoperiod. Temperatures in the green-house were maintained at between 20 and 24°C. Eachcontainer was fitted with a floating Perspex “raft” thatheld up to 32 plants. Unused plugs in the raft andthe rest of the container were covered with blackpolythene to prevent algal growth. The hydroponicssolution was a modified 1/10th strength Johnson’s so-lution [27] supplemented with either 250 μM Ca(NO3)2 or (NH4)2(SO4). Final nutrient composition,excluding nitrogen, was: 200 μM KH2PO4, 200 μMK2SO4, 100 μM MgSO4, 100 μM CaSO4, andmicronutrients: 5 μM Cl, 2.5 μM B, 0.2 μM Mn,0.2 μM Zn, 0.1 μM Mo, 0.05 μM Cu, and 50 μM Fe2+.Containers were fitted with 20 L per minute centrifugalpumps, to circulate and aerate media. Solutions weremonitored periodically for oxygen levels, pH andtemperature. Powdered calcium carbonate (CaCO3)was added to buffer pH in the range of 6–7.5. MediaNH4+ and NO3- concentrations were assayed using theKalcsits and Guy Plant Methods 2013, 9:27 Page 6 of 9http://www.plantmethods.com/content/9/1/27phenol: hypochlorite [28] and perchloric acid [29]methods, respectively. The solution was completelyreplaced every 14 days to ensure that there was nosubstantial decrease (>10%) in concentration of nitrateor ammonium over time that could increase the solu-tion δ15N.Sampling and natural abundance isotope analysisAfter 45 days of growth, plants were separated intoleaves, stems, roots and the original cutting. Sampleswere flash frozen in liquid nitrogen and stored at −80°Cuntil samples could be freeze-dried at −50°C for twodays. Once dried, roots, leaves, stems, cuttings and thecuttings collected at the start of the experiment wereweighed. Samples were then ground to a fine powderusing a mortar and pestle and then a ball mill (FritschLaborgeratebau, Terochem Scientific). Subsamples of3 ± 0.1 mg were weighed into tin capsules (ElementalMicroanalysis Ltd., 8×5 mm, D1008) and analyzed fornitrogen concentration and δ15N on a PDZ EuropaANCA-GSL elemental analyzer interfaced to a PDZEuropa 20–20 isotope ratio mass spectrometer(Sercon Ltd., Cheshire, UK) (University of CaliforniaStable Isotope Facility, Davis, CA). Isotopic compos-ition is expressed as δ15N:δ15N ¼ RsampleRstandard−1  1000 ð1Þwhere, Rsample is the15 N/14 N isotope ratio of thesample and Rstandard is the isotope ratio of a knownstandard (air). The δ15N values of the ammonium andnitrate salts used for the growth media were −0.96and +58.5‰, respectively.Correcting for carried-over pre-existing cutting nitrogenin growing plant organsThe δ15N and nitrogen concentration of the cutting afterharvest was compared to the δ15N and nitrogen concen-tration of the cutting before the start of the experimentto correct for pre-existing nitrogen remobilized to theactively growing plant. Nitrogen content of the cuttingat harvest (Nend) can be calculated as:Nend ¼ Biomasscutting end  N½ cutting end ð2Þwhere, Biomasscutting endand [N]cutting end are the bio-mass and nitrogen concentration of the cutting atharvest. Since the cutting was part of the growingstem, dry mass accumulated in the cutting. The initialdry mass of the cutting (Biomasscutting start) was esti-mated from the fresh mass of the cutting at the startof the experiment and the mean dry mass content ofthe cuttings sampled at the start of the experiment(0.584). From this, nitrogen content at the start ofthe experiment (Nstart) was estimated as:Nstart ¼ Biomasscutting start  N½ start ð3Þwhere, [N]start is the mean nitrogen concentration of thecuttings at the start of the experiment. Assuming that theisotopic composition of a cutting at harvest (δ15Nend) is amixture of stem δ15N (δ15Nstem) (containing a portion ofremobilized nitrogen) and non-remobilized nitrogenremaining in the cutting (δ15Nstart), the fraction of non-remobilized (fnonremobilized) nitrogen can be estimated as:f nonremobilized ¼δ15Nend−δ15Nstem δ15Nstart−δ15Nstem  ð4ÞThe amount of nitrogen remobilized (Nremobilized) tonew growth can then be calculated as:Nremobilized ¼ Nstart− Nend  f nonremobilized  ð5ÞThe proportion of carried-over pre-existing nitrogenin the general plant nitrogen pool that was carried overinto new growth (C) is given by:C ¼ NremobilizedNplantð6Þwhere, Nplant is equal to the sum of all nitrogen contentsin the roots, leaves and stems (including new andremobilized nitrogen allocated to the expanded cutting):Nplant ¼ Nleaves þNroots þNstem þNend 1−f nonremobilized  ð7ÞAssuming that there is a proportionate distribution ofremobilized nitrogen throughout the plant (i.e., C is thesame for all plant organs), the mass balance equationshowing the contributions of new and carry-over nitro-gen to measured δ15N for roots, stems and leaves is:δ15Nunadjusted ¼ δ15Ncarry−over  C þ δ15Nnew 1−Cð Þ ð8Þwhere, δ15Nunadjusted, δ15Ncarry-over and δ15Nnew are themeasured isotopic compositions of each plant organ, theisotopic composition of remobilized nitrogen (equal toδ15Nstart), and the isotopic composition of newly assimi-lated nitrogen, respectively. Equation 8 can be rearrangedto yield:δ15Nnew ¼ δ15Nmeasured−δ15Ncarry−over  C1−Cð Þ ð9ÞThis equation was used to obtain root, stem andleafδ15N values adjusted for the contribution of carry-over nitrogen from the cuttings.Kalcsits and Guy Plant Methods 2013, 9:27 Page 7 of 9http://www.plantmethods.com/content/9/1/27Statistical analysisTwo-way ANOVA was used to test for the fixed effectsof nitrogen source and genotype on (1) biomass, (2)measured δ15N values, (3) the proportion of cuttingnitrogen remobilized to the growing plant, (4) the pro-portion of nitrogen in new growth that is carry-overnitrogen, and (5) the adjusted δ15N values of plant, rootand leaf tissues. The statistical model was as follows:Y ij ¼ μþ αi þ τj þ βij ð10Þwhere, μ is the overall mean response, αi is the effectdue to the genotype, τj is the effect due to the nitrogensource and βij is the effect due to any interaction be-tween the genotype and nitrogen source. Analysis ofvariance procedure was carried out using GraphpadPrism 6 (La Jolla, CA, USA) to obtain estimates of themeans followed by Tukey’s multiple comparison tests toseparate means. Differences between treatments de-scribed as significant are those where P <0.05. Wholeplant δ15N was plotted against biomass and per centcarry-over nitrogen to examine the influence on mea-sured δ15N of genotypes grown using nitrate or ammo-nium. Non-linear regression was performed for themeasured plant δ15N versus biomass using GraphpadPrism 6 to fit an exponential model to the data. Linearregression was performed on adjusted plant δ15N versusbiomass and on both measured and adjusted plant δ15Nversus the proportion of carry-over nitrogen in newplant growth.Flushing vegetative cuttings without addition ofexogenous nitrogenVegetative cuttings from four randomly selected P.trichocarpa genotypes were selected from a commongarden experiment and stored at 2°C until flushing.Complementary cuttings (N = 5) were selected whereone was allocated for flushing and the cutting immedi-ately adjacent was separated into bud and cutting stemand oven-dried at 60°C for four days. Cuttings allocatedfor flushing were randomly placed in a floating foam raftin ddH2O for 21 days. At harvest, plants were separatedinto cutting stem, shoot and roots and oven dried for4 days at 60°C. Samples were then weighed, ground andanalyzed for nitrogen content and isotope compositionas described above. Samples were then analyzed usingan Isoprime (GV Instruments) Isotope Ratio MassSpectrometer (IRMS) coupled with an ElementarVario EL Cube Elemental Analyzer (EA) (UBC Facultyof Forestry Stable Isotope Facility). t-tests were usedto compare nitrogen content and isotope means be-tween genotypes and plant parts (P = 0.05).Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsLAK conceived and carried out the nitrogen isotope discriminationexperiments, developed the correction and drafted the manuscript. RDGparticipated in its design and helped to draft the manuscript. All authorsread and approved the final manuscript.AcknowledgementsThis work was funded by a Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) Discovery Grant to RDG. LAK was supported by aNSERC Vanier Canada Graduate Scholarship. Thank you to the AESB-AAFCAgroforestry Development Centre for provision of plant material from theAgCanBap balsam poplar collection. Thank you to two anonymous reviewersfor their valuable comments to improve upon this manuscript. Appreciationis extended to Rob Johnstone, Limin Liao and Don Reynard for technicalassistance.Received: 25 February 2013 Accepted: 1 July 2013Published: 12 July 2013References1. Comstock JP: Steady-state isotopic fractionation in branched pathwaysusing plant uptake of NO3- as an example. Planta 2001, 214:220–234.2. Evans RD: Physiological mechanisms influencing nitrogen isotopecomposition. Trends Plant Sci 2001, 6:121–126.3. Dawson TE, Mambelli S, Plamboeck AH, Templer PH, Tu KP: Stable isotopesin plant ecology. Ann Rev Ecol Sys 2002, 33:507–559.4. Pritchard ES, Guy RD: Nitrogen isotope discrimination in white spruce fedwith low concentrations of ammonium and nitrate. Trees-Struct Funct2005, 19:89–98.5. Houlton BZ, Sigman DM, Schuur EAG, Hedin LO: A climate driven switch inplant nitrogen acquisition within tropical forest communities. P Natl AcadSci USA 2007, 104:8902–8906.6. 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Analyst 1967, 92:311–315.doi:10.1186/1746-4811-9-27Cite this article as: Kalcsits and Guy: Quantifying remobilization of pre-existing nitrogen from cuttings to new growth of woody plants using15N at natural abundance. Plant Methods 2013 9:27.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitKalcsits and Guy Plant Methods 2013, 9:27 Page 9 of 9http://www.plantmethods.com/content/9/1/27

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