Plant and Soil222:119–137,2000.
©2000Kluwer Academic Publishers.Printed in the Netherlands.
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Foliar free polyamine and inorganic ion content in relation to soil and soil solution chemistry in two fertilized forest stands at the Harvard Forest, Massachusetts
Rakesh Minocha1,∗,Stephanie Long1,Alison H.Magill2,John Aber2and William H. McDowell3
deposition1USDA Forest Service,Northeastern Research Station,P.O.Box640,Durham,NH03824,USA;2Complex Systems Research Center,University of New Hampshire,Durham,NH03824,USA and3Department of Natural Resources, University of New Hampshire,Durham,NH03824,USA
Received24September1999.Accepted in revised form29February2000
Key words:ammonium nitrate,calcium,Harvard Forest,magnesium,nitrate leaching,polyamines
Abstract
Polyamines(putrescine,spermidine,and spermine)are low molecular weight,open-chained,organic polycations which are found in all organisms and have been linked with stress responses in plants.The objectives of our study were to investigate the effects of chronic N additions to pine and hardwood stands at Harvard Forest,Petersham, MA on foliar polyamine and inorganic ion contents as well as soil and soil solution chemistry.Four treatment plots were established within each stand in1988:control,low N(50kg N ha−1yr−1as NH4NO3),low N+sulfur(74kg S ha−1yr−1as Na2SO4),and high N(150kg N ha−1yr−1as NH4NO3).All samples were analyzed for inorganic elements;foliage samples were also analyzed for polyamines and total N.In the pine stand putrescine and total N levels in the foliage were significantly higher for all N treatments as compared to the control plot.Total N content was positively correlated with polyamines in the needles(P≤0.05).Both putrescine and N contents were also negatively correlated with most exchangeable cations and total elements in organic soil horizons and positively correlated with Ca and Mg in the soil solution(P≤0.05).In the hardwood stand,putrescine and total N levels in the foliage were significantly higher for the high N treatment only as compared to the control plot.Here also, total foliar N content was positively correlated with polyamines(P≤0.05).Unlike the case with the pine stand,in the hardwood stand foliar polyamines and N were significantly and negatively correlated with foliar total Ca,Mg, and Mn(P≤0.05).Additional significant(P≤0.05)relationships in hardwoods included:negative correlations b
etween foliar polyamines and N content to exchangeable K and P and total P in the organic soil horizon;and positive correlations between foliar polyamines and N content to Mg in soil solution.With few exceptions,low N +S treatment had effects similar to the ones observed with low N alone for both stands.The changes observed in the pine stand for polyamine metabolism,N uptake,and element leaching from the soil into the soil solution in all treatment plots provide additional evidence that the pine stand is more nitrogen saturated than the hardwood stand. These results also indicate that the long-term addition of N to these stands has species specific and/or site specific effects that may in part be explained by the different land use histories of the two stands.
Abbreviations:Perchloric acid(PCA),Polyamines(PAs),Zero tension lysimeter(ZTL),hardwoods(HW)
Introduction
There is increasing concern about the potential ad-verse effects of elevated rates of N deposition on water ∗FAX No:6038687604.E-mail:rminocha@hopper.unh.edu quality and the health of forest ecosystems(Aber et al.,1989,1998;Rasmussen and Wright,1998).This concern stems from the fact that in1990the United States(US)Clean Air Act targeted a50%reduction in S deposition but only a10%reduction in N depos-
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ition(McNulty et al.,1996).Although most temperate forests are N limited,continuous deposition of N from the atmosphere can move them towards nitrogen sat-uration.Nitrogen saturation has been defined as the availability of ammonium and nitrate in excess of total combined plant and microbial nutritional demand (Aber et al.,1989).It is important to understand how chronic additions of N to ecosystems change the struc-ture and function of forest ecosystems(Asner et al., 1997;Jefferies and Maron,1997).
Long-term elevated N deposition typically leads to an increase in the concentration of total foliar N, with or without similar changes in the important base elements such as Ca,Mg and K(Aber et al.,1995; Magill et al.,1997,1999;Rasmussen and Wright, 1998;van Dijk and Roelofs,1988).This increase in leaf N content also leads to significant shifts in the internal partitioning of N within the leaf.For example in conifers,N deposition significantly increases leaf N present in the form of free amino acids such as arginine (Ericsson et al.,1993,1995;Näsholm et al.,1997). Little is known about N partitioning for hardwoods under these conditions.Lawlor(1992)suggested that these changes in N partitioning are probably not re-lated to leaf function.This idea,however,has yet to be experimentally tested in terms of whether the alter-ations in N partitioning due to long-term N deposition actually hav
e a positive or a negative effect on photo-synthetic capacity and biomass production.A possible decoupling of the relationship between foliar N and photosynthetic rate may occur under these conditions.
The aliphatic polyamines(putrescine,spermidine, and spermine)play an important role in the growth and development of all living organisms.They are meta-bolically derived from the amino acids arginine and ornithine,and at cellular pH they carry a net positive charge(Cohen,1998).Abiotic stress conditions such as low pH,high SO2,high salinity,osmotic shock, nutrient stress,low temperature(Flores,1991and ref-erences therein)and high Al(Minocha et al.,1992, 1996)all result in an increase in cellular putrescine levels.Polyamines show a reverse proportionality to cellular elements such as Ca,Mg,Mn,and K in re-sponse to Al treatment in periwinkle(Catharanthus roseus)and red spruce(Picea rubens)cells(Minocha et al.1992,1996;Zhou et al.,1995).A key distinction between the polyamines(organic cations)and the inor-ganic elements is that,even if the Ca2+and Mg2+)undergo recompartmentalization in response to external stimuli,their cellular levels are derived mainly from uptake across the biological membranes and this uptake ultimately depends upon their availab-ility in the soil or soil solution.In contrast,polyamines are synthesized within the cell,permitting adjustment of their cellular concentrations to meet physiological
needs.Also,cellular polyamine levels can be regu-lated by conjugation,degradation,and sequestration via enzymatic means(Minocha et al.,1996).Thus polyamine synthesis may play an important role in the survival of plants under stress(Galston,1989).Recent work examining the concentration of polyamines in plant foliage has been aimed at using foliar polyam-ine concentrations as indicators of stress(Dohmen et al.,1990;Minocha et al.,1997;Santerre et al.,1990). In the case of mature red spruce trees,an increase in foliar putrescine concentration was associated with a decrease in foliar and soil Ca and Mg concentrations and an increase in the Oa horizon soil Al or Al:Ca ratios(Minocha et al.,1997).
Structurally,polyamines are composed of carbon, hydrogen,and nitrogen.Therefore,we suspect that their levels may also change in response to chronic N additions,thus affecting the internal N partitioning within the leaf,a situation similar to that observed with free amino acids in conifers.The objective of this study was to determine the effects of chronic additions of N on:(1)foliar polyamines(a proposed stress in-dicator)and inorganic ions;(2)soil and soil solution inorganic ion chemistry,especially Al mobilization; and(3)the relationship between polyamines and soil chemistry in pine and hardwood stands.
Materials and methods
Study sites
The study plots are located at Harvard Forest,Peter-sham,MA(42◦30 N,72◦10 W).This site is a part of the National Science Foundation’s Long-Term Eco-logical Research(LTER)program.These plots are a part of the ongoing study on chronic nitrogen addi-tions since1988(Aber et al.,1993;Magill et al.,1997, 1999).An even-aged red sinosa)stand,74 yr old,and an adjacent mixed hardwood stand,approx. 55yr old,were chosen for this study.The hardwood stand is dominated by black oak(Quercus vetulina Lam.)and red oak(Q.Rubra Michx.f.,respectively) with significant amounts of black birch(Belutinaa lenta L.),red maple(Acer rubrum L.),and American beech(Fagus grandifolia Ehrh.).Most of the currently forested area at this site was in cultivation or pas-tureland during the mid-1800’s(Foster,1992).The
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dominant soil types are stony sandy loams classified as Typic Dystrochrepts.Mean annual temperature ranges from19◦C in July to–12◦C in January and mean total annual precipitation is112cm.The estimated nitrogen deposition to the forest is about6kg ha−1yr−1wet and2kg ha−1yr−1dry(Aber et al.,1993;Magill et al.,1997;Ollinger et al.,1993).
Treatments
Four treatment plots were established within each stand:control,low N,low N+sulfur(N+S),and high N.Each plot measured30×30m(0.09ha)and was divided into36subplots(each5×5m).Fertilizer additions of NH4NO3and Na2SO4began in1988as six equal applications over the growing season.The fertilizer was weighed,mixed with20L of water (equivalent to0.002cm rainfall)and applied using a backpack sprayer.Two passes were made across each plot to ensure an even distribution of fertilizer.
As described in Magill et al.(1997),a partial ap-plication was made in year1(1988).The total amount of fertilizer applied was38kg N ha−1yr−1to the low nitrogen treatment and the nitrogen portion of the N+S treatment,113kg N ha−1yr−1to the high nitrogen treatment,and74kg(S)ha−1yr−1to the N+S plots. Applications for all following years were at the rate of 50kg N ha−1yr−1to the low and N+S plots and150 kg N ha−1yr−1to the high N plots.Sulfur additions remained the same as used for year one.
Collection and analyses of needle samples
Foliage samples were collected during thefirst week of August each year from mid to upper canopy b
ranches of dominant or co-dominant trees using a shotgun.Early August was chosen as the sampling time because the trees were still physiologically very active at this time,compared to early or late summer. At each sampling time,current-year needle samples were collected from20different red pine trees in the pine stand and leaves from10different trees of black or red oak and red maple each were collected from the hardwood plots.A sub-sample was taken from each in-dividual tree collection for analyses of polyamine and exchangeable inorganic elements.The remaining pine needle samples from20different trees in each plot were pooled into5composite samples of4trees per sample for total inorganic elements and N analyses. Similarly,the remaining samples from10–12trees per species collected from each plot in the hardwood stand were pooled into4composite samples.Red and black oak were treated as a single species in all collections. Total elements and nitrogen analyses
The composite samples were dried at70◦C for48 h and ground using a Wiley mill with a1mm mesh screen.The ground samples were dried overnight at 70◦C and analyzed for N content using near-infrared (NIR)spectroscopy(Bolster et al.,1996;McLel-lan et al.,1991).These samples were also used for extraction of total inorganic ion content by a modi-fication of the method of Isaac and Johnson(1976) as described in Minocha and Shortle(1993).The ex-tracts were analyzed for total Ca,Mg,Mn,K,Al, and P content using a Beckman Spectrospan V ARL DCP(Direct Current Plasma E
mission Spectrometer, Beckman Instruments,Inc.,Fullerton,CA)using the Environmental Protection Agency’s method number 66-AE0029(1986).
Analysis of polyamines and exchangeable inorganic elements
The fresh foliage samples were placed in individual pre-weighed microfuge tubes containing1ml of5% perchloric acid(PCA).The tubes were kept on ice dur-ing transportation to the laboratory and then stored at –20◦C until they were processed.The samples were weighed,frozen and thawed(3X),and centrifuged at 13,000rpm in a microfuge for10min.Details of the freeze-thawing extraction procedure are described in Minocha et al.(1994).The freeze-thawing method was also chosen for the extraction of exchangeable fraction of inorganic ions.This method extracted a consistent fraction of the total acid extractable inor-ganic ions from foliage of various species of mature trees and the quantity of this fraction varied for each ion type and tree species(Table1and Minocha et al.,1994).The moisture content data for individual exchangeable ion samples was not collected.There-fore,for comparison of total and exchangeable ions on weight basis,an average moisture content of53%, 62%,and61%has been used for this site from data collected in1992for pine(n=19),oak(n=33),and maple(n=45),respectively to convert fresh weight to dry weight.
Briefly,the samples for both inorganic ions as well as for polyamines were frozen at–20◦C and thawed at room temperature,repeating the process two more times.Duration of the freezing step could vary from4
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Table1.Comparison of effects of nitrogen treatments on total and exchangeable inorganic ion levels in the foliage of pine,oak and maple trees.Data has been averaged over three year period(1995–1997).Data for exchangeable ions are mean±se of n=60for pine stand and n=30for hardwoods.Data for total ions are mean±se of n=15for pine stand and n=12for hardwoods.The numbers in parenthesis indicate%of total ions extracted as exchangeable.The moisture content data for individual exchangeable ion samples was not collected.Therefore,for comparison of total and exchangeable ions on weight basis,an average moisture content of53%,62%,and61%has been used for this site from data collected in1992for pine(n=19),oak(n=33),and maple(n=45),respectively to convert FW to DW
Treatment Ion Inorganic ions Pine(µmol g−1DW)Oak(µmol g−1DW)Maple(µmol g−1DW)
Control Ca Total62.5±4.8121.5±5.9156.0±6.8
Low N72.3±5.1113.5±8.1137.0±6.7
High N60.0±5.571.8±6.2116.4±6.1
Control Exchangeable11.4±0.6(18.3%)102.1±5.9(84.0%)79.2±4.7(50.8%)
Low N11.9±0.7(16.5%)87.1±4.7(76.7%)69.7±2.3(50.9%)
High N9.9±0.9(16.5%)57.3±5.0(79.9%)59.2±3.4(50.9%)
Control Mg Total32.4±1.562.8±2.164.7±3.3
Low N35.5±1.363.1±2.359.9±1.3
High N35.4±1.354.1±3.355.7±2.9
Control Exchangeable13.6±0.5(41.9%)56.1±3.4(89.3%)43.7±2.3(67.5%)
Low N14.7±0.4(41.3%)58.0±3.4(91.9%)43.5±1.4(72.7%)
High N13.5±0.4(38.1%)51.0±3.0(94.2%)38.5±1.9(69.1%)
Control Mn Total16.7±1.141.6±2.337.4±2.3
Low N22.4±2.127.3±2.023.6±0.9
High N15.8±1.615.3±0.922.5±1.9
Control Exchangeable  3.6±0.2(21.3%)50.8±3.5(122.1%)∗21.1±1.5(56.6%)
Low N  3.9±0.3(17.3%)33.4±2.4(122.4%)∗14.5±1.0(61.6%)
High N  3.2±0.4(20.5%)17.4±1.5(113.4%)∗11.1±0.9(49.5%)
Control K Total120.1±6.7218.7±9.5184.6±11.8
Low N125.5±7.2265.7±12.0171.6±10.5
High N135.3±7.4202.2±9.4176.0±9.7
Control Exchangeable72.8±3.8(60.6%)158.0±6.8(72.3%)132.2±6.0(71.6%)
Low N79.6±3.6(63.4%)165.1±7.2(62.2%)138.8±5.9(80.9%)
High N84.0±3.2(62.1%)157.5±6.7(77.9%)147.0±5.8(83.5%)
∗The calculation of greater than100%extraction for Mn in the case of oak was possibly caused by slight overestimation of the mean moisture content for1995–1997.
h to a few days.Samples were allowed to thaw com-pletely(approximate time1–1.5h)before refreezing. After freeze-thawing,the samples were centrifuged at 13,000×g.This supernatant was used directly for free polyamine analysis without further dilution and for inorganic-ion analysis after proper dilution with distilled,deionized water(final concentration of PCA 0.01or0.02N)by the procedures described below. The diluted fractions were analyzed for inorganic ion content with a Beckman Spectrospan V ARL DCP as described above.For quantitation of polyamines, heptanediamine was added as an internal standard to aliquots of the above extracts prior to dansylation. Fifty or one hundredµL of the extract were dansylated according to the procedure described in Minocha et al.(1990).Dansylated polyamines were separated by reversed phase HPLC(Perkin-Elmer Corp.,Norwalk,CT)using a gradient of acetonitrile and heptane-sulfonate,and quantified by afluorescence detector (Minocha et al.,1990).
Collection and analyses of soil and soil solution samples
Three sets of two adjacent soil cores(<30cm apart) were taken to a depth of10cm in the mineral soil in each of the three designated subplots(nine samples per plot).Cores were split into organic(Oe+Oa)and min-eral horizons(top10cm)and placed in gas-permeable polyethylene bags.The soils were air-dried,sieved (<2mm size),and stored at room temperature prior to analyses.Before analyses,the mineral soil and organic soil samples were oven-dried at105◦C and70◦C, respectively.
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Exchangeable inorganic elements were determined from a sub-fraction of the above samples using a modi-fication of the procedure of Taylor(1987).Briefly, either6g of mineral soil or1g of organic soil was added to30ml of extraction solution(0.05N HCl and 0.025N H2SO4)and placed on a gyratory shaker at90 rpm for15min.The extract wasfiltered with a glass fiber syringefilter(Gelman A/E,Gelman Sciences, Ann Abror,MI)and stored at4◦C until quantitation by DCP.
Prior to digestion for total elemental analysis,a sub-sample of air-dried and sieved soil was processed for1min in a Shatter Box Laboratory Mill(Spex Industries,Inc.,Edison,NJ)to powder the sample. Microwave digestion(Hallett and Hornbeck,1997) was used for obtaining inorganic elements.Briefly, for mineral soils,0.1g sample was digested with5 ml of concentrated HNO3,2ml of c
oncentrated HCl, 2ml offluoroboric acid(HBF4)and2ml of H2O2. For organic soils,0.1g sample was digested the same way with acids but without the presence of H2O2.The following microwave programs were used.Given in pairs are:Time(min)–Power(Watts).For mineral soil, 1–250,1–0,5–250,5–400,5–500,and5–600.For organic soil,1–250,1–0,5–250,5–400,1–0,5–500, 1–0,5–600,1–0,and2.5–650.Total running time was 27.5min.
The details on installation of zero tension lysi-meters(ZTL)and soil solution sample collection are described in Currie et al.(1996)and McDowell et al. (1998).Briefly,5polyethylene ZTL’s were installed per plot except for N+S plots.Solutions were collected after major rain events and all5samples per plot were pooled prior to analyses.Over the course of three years (1995–1997),the collections were made approx.50 times from each plot.Samples were transported on ice to the laboratory andfiltered through pre-combusted Whatman GF/F glassfiberfilters(Whatman Inc., Clifton,NJ)within36h of collection before freezing. These solutions were analyzed for inorganic elements using DCP as described earlier.
Statistical methods
Linear regression analyses were performed to es-tablish the strength and significance of relationship
s between two different variables(n=12except for soil samples where n=8)using Excel5.0for Windows(Mi-crosoft Corporation,Roselle,IL).Data for each ,foliar or soil Ca,Mg and Al)were analyzed as a series of one-way analysis of variance(ANOV A)to determine whether statistically significant differ-ences occurred between control and treatment plots for each individual variable.When F values for one-way ANOV A were significant,differences in treatment means were tested by Tukey’s multiple comparis-ons test.ANOV A and Tukey’s tests were performed with Systat for Windows,version7.01(SYSTAT Inc., Evanston,Il)and a probability level of0.05was used for tests unless specified otherwise.
Results
With few exceptions,the low N+S treatment showed effects similar to the ones observed for low N treat-ment alone for both stands.For this reason,results for low N+S will not be discussed separately.Also,in foliage exchangeable ions always represented a con-sistent fraction of the total ions.Even though the quantity of this fraction varied for each ion type and tree species,nitrogen treatments had similar effects on both exchangeable and total Ca,Mg,Mn,and K levels in each case for all three species(Table1).Thus due to this similarity of trends between exchangeable and total ions,only total inorganic ion data are used for further comparison of foliar results with soil and soil sol
ution data.
Foliar polyamines and N content
Pine:There was a significant increase in the level of putrescine in the needles of trees growing on all three treatment plots as compared to the control plot (Figure1A–D).A small but statistically significant increase was also observed in spermidine levels in response to high N treatment.Spermine,which was a relatively small proportion of free polyamines in red pine,increased significantly in response to low and high N additions.In spite of year to year vari-ations in the total amounts of polyamines(possibly due to different growth conditions resulting from vari-able weather patterns),similar trends were observed for each of the three years of data collection. Hardwoods:Putrescine levels in oak leaves were three-to four-fold higher in response to high N treat-ment for each of the three years of this study(Fig-ure2A–D).For other treatments no significant change was observed.The level of spermidine and spermine did not change significantly in response to low and high N treatments.While there were annual variations

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