Soil Biology amp Biochemistry 41 (2009) 1210-1220

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Soil Biology amp Biochemistry

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Soil amino acid composition across a boreal forest successional sequenceNancy R Werdin-Pfisterer Knut Kiellan Richard D BooneDepartment of Biology amp Wildlife Institute of Arctic Biology University of Alaska Fairbanks Fairbanks AK 99775 USA

ARTICLE INFO ABSTRACT

Article historyReceived 2 January 2009Received in revised form27 February 2009Accepted 6 March 2009Available online 2 April 2009

Soil amino acids are important sources of organic nitrogen for plant nutrition yet few studies haveexamined which amino acids are most prevalent in the soil In this study we examined the compositionconcentration and seasonal patterns of soil amino acids across a primary successional sequenceencompassing a natural gradient of plant productivity and soil physicochemical characteristics Soil wascollected from five stages (willow alder balsam poplar white spruce and black spruce) of the floodplainsuccessional sequence on the Tanana River in interior Alaska Water-extractable amino acid compositionand concentration were determined by HPLC Irrespective of successional stage the amino acid pool wasdominated by glutamic acid glutamine aspartic acid asparagine alanine and histidine These six aminoacids accounted for approximately 80 of the total amino acid pool Amino acid concentrations were anorder of magnitude higher in coniferous-dominated late successional stages than in early deciduous-dominated stages The composition and concentration of amino acids were generally constantthroughout the growing season The similar amino acid composition across the successional sequencesuggests that amino acids originate from a common source or through similar biochemical processesThese results demonstrate that amino acids are important components of the biogeochemical diversity ofnitrogen forms in boreal forests

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KeywordsAmino acidsBoreal forestNitrogen cycleOrganic nitrogenSuccession

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1 Introduction

The role of dissolved organic nitrogen (N) in meeting the nutri-tional requirements of forest and agricultural plants recently hasreceived much attention There is a worldwide geographic distri-bution of plant species and growth forms that use organic N in theform of amino acids peptides and proteins directly through rootswithout mycorrhizae or via ericoid ecto- or other mycorrhizalassociations (Kitelland 2001 Lipson and Nasholrn 2001) Numerouslaboratory in situ and field studies have shown that plants candirectly absorb organic forms of N namely free amino acids therebycircumventing the traditional mineralization bottleneck (Chapinet al1993 Kielland et al 1994 Raab et al 1996 Schimel and Chapin1996 Nasholm et al 1998 Nordin et al 2001) Inferences regardingthe importance of amino acids as an organic N source have beenlargely based on physiological studies of plant uptake few studieshave examined the free amino acid composition of the soil

Dissolved organic N may be especially significant in borealregions where N mineralization and bulk organic matter decom-position predominantly are slow due to a cold dry climate (VanCleve and Alexander 1981 Flanagan and Van Cleve 1983) Van

Corresponding author Tel +1 1 907-474-7164E-mail addressffekallafedu (K Kielland)

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Cleve et al 1991) Organic forms of N dominate over inorganic Nforms in the Tanana River floodplain soils in interior Alaska (Kiel land et al 2007) In addition the boreal forests of interior Alaskareceive minimal N in the form of atmospheric deposition(058 kg N ha-1 y-1 in 2001 NADP 2002) The Tanana River flood-plain successional sequence accordingly provided an ideal settingto study the soil amino acid composition of a relatively pristineorganic N-dominated ecosystem

Primary succession on the Tanana River occurs over a timescaleof more than 300 years (Viereck et al 1993a) and is well docu-mented by over three decades of research (Van Cleve et al1993b)The successional sequence represents a natural gradient of plantproductivity and soil physicochemical characteristics including soiltemperature organic matter content pH carbon and N concen-tration and decomposition rates (Van Cleve et al 1993a C Vierecket al1993a Yarie et al 1993) Along the successional sequencethere are several critical turning points that result in majorfunctional changes to the ecosystem 1) canopy closure andincreased litter deposition promotes forest floor development 2)addition of the N-fixing species thinleaf alder (Alnus incanasubsp tenuifolia) increases soil N 3) forest functional type shiftsfrom shrub to tree dominance 4) overstory composition changesfrom deciduous to coniferous species and 5) development of aninsulating moss layer reduces soil temperature and promotes theformation and maintenance of permafrost (Viereck 1989) These

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220

vegetation soil and ecosystem changes with succession may affectthe composition and concentration of soil amino acids across thissuccessional sequence

Soil amino acid concentrations have been shown to vary overthe growing season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005) and likely reflectseasonal changes in soil and plant biology Increased uptake ofavailable soil amino acids during plant and microorganism growthcycles may deplete the amino acid concentrations in the soil (Joneset al 2005b Weintraub and Schimel 2005) Seasonal freeze-thawand dry-rewet events may cause flushes of amino acids from lysisof microbial mycorrhizal or root tissue (Lipson and Monson 1998)as well as the physical disintegration of soil organic matter struc-ture (Ivarson and Sowden 1966) Seasonal changes in microbialactivity may alter microbial production of exoenzymes that breakdown proteins into soluble peptides and amino acid constituents(Abuarghub and Read 1988 Lipson and Niisholm 2001 Read andPerez-Moreno 2003) Other relevant seasonal amino acid sourcesmay include turnover of roots and mycorrhizae (Ruess et al 2006)root exudation (Jones 1999 Jones et al 2005b) and litter inputs(Chapin and Kedrowski 1983 Chapin et al1986) Although thebody of work on soil amino acids including their temporal changesand the causes for those changes has grown rapidly in the pastdecade fundamental information on the composition and seasonaldynamics of amino acids is still very limited

Our research quantified the composition temporal and land-scape patterns of free amino acids in the soils of the Tanana Riversuccessional sequence in interior Alaska We hypothesized that thecomposition of the soil amino acid pool would vary across thesuccessional sequence with amino acids with simple molecularstructures (eg glycine aspartic and glutamic acid) prevalent in theearly deciduous-dominated successional stages with younger andless chemically complex organic matter and amino acids witha more complex molecular structure (eg arginine histidinephenylalanine) common in the later coniferous-dominatedsuccessional stages with an older and larger reservoir of chemicallycomplex and recalcitrant organic matter Second we hypothesizedthat the concentration of soil amino acids would increase across thesuccessional sequence as plant-derived organic matter accumu-lated and N mineralization and microbial immobilization of aminoacids slowed with reduced soil temperatures and more recalcitrantsoil organic matter Finally we hypothesized that soil amino acidconcentration would be highest in the spring because of root andmicrobial lysis over winter To test these hypotheses we investi-gated the composition of the soil amino acid pool the concentra-tion of soil amino acids and the seasonal dynamics of soil aminoacids across a primary successional sequence on the Tanana Riverfloodplain in interior Alaska To our knowledge this is one of thefirst studies to examine the full suite of possible amino acids acrossa primary forest successional sequence

2 Materials and methods

21 Study sites

The study area was located on the Tanana River floodplainwithin the Bonanza Creek Long Term Ecological Research (LTER)site 20 km southwest of Fairbanks in interior Alaska (lat 64deg44Nlong 148o15W elevation 120 m) The climate is strongly conti-nental and semiarid with cold winters and warm dry summers(Viereck et al 1993b) Mean annual temperature is -33degC withtemperature extremes ranging from -50 oC in winter to 35degC insummer Mean annual precipitation is 269 mmy-1 with approxi-mately 37 falling as snow The growing season is 100 days or lessThe Tanana River nearly 1000 km in length (Collins 1990) is the

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largest tributary of the Yukon River and carries a heavy silt loadwith 85 of its discharge from glacier-fed tributaries originating inthe Alaska Range (Anderson 1970) The Tanana River floodsfrequently due to snowmelt in spring anci increased rainfall andglacial meltwater in late summer thereby producing alluviumterraces of progressively greater age with increasing distance fromthe rivers edge (Van Cleve et al 1993a yarie et al 1998)

The study sites comprising a primary successional sequencethat encompasses a natural gradient of terrace age plant produc-tivity and soil physicochemical characteristics (Table 1) c included five major successional stages willow alder balsam poplar whitespruce and black spruce We established three replicate 900 m2

plots (each 30 x 30 m or 20 x 45 m) in each of the five successionalstages for a total of 15 plots located along an 11 km stretch of theTanana River Replicated plots of each successional stage hada similar vegetative community stand structure and age

22 Vegetation

We conducted a vegetation inventory at each plot to charac-terize the general vegetation composition of the five successionalstages Willow plots were characterized by the dominance of SalixL species (Salix alaxensis (Anderss) Coville Salix niphoclada RydbSalix interior Rowlee Salix lucida subsp lasiandra (Benth) E Murrand Salix pseudomyrsinites Anderss) Populus balsamifum L seed-lings and scattered clumps of A incana subsp tenuifolia (Nutt)Breitung the understory was dominated by Equisetum variegatumSchleich ex FWeber amp D Mohr and Equisetum arvense L Alder plotswere characterized by a dense canopy of A incana subsp tenuifoliascattered Spseudomyrsinites shrubs and P balsamifera saplings andtrees the understory was dominated by E arvense and Chamerionangustifolium (L) Holub Balsam poplar plots were characterized bya tall mature P balsamifera overstory and a dense shrub layer of Aincana subsp tenuifolia the understory was fairly sparse due to thehigh litter cover White spruce plots were characterized by a largemature Picea glauco (Moench) Voss overstory and a dense tall shrublayer of Rosa acicularis Lindl Alnus viridis subsp fruticosa (Rupr)Nyman A incana subsp tenuifolia and Viburnum edlue (Michx)Raf the understory was dominated by a thick feather moss layer ofHylocomium splendens (Hedw) Schimp Black spruce plots werecharacterized by an open canopy of mature Picea mariana (Mill)BSP trees a diverse tall shrub layer of R acicularis A viridis subspfruticosa A incana subsp tenuifolia Salix arbusculoides Anderss Salaxensis Betula glandulosa Michx and Betuta nana L the under-story was dominated by a dense low shrub layer of Ledum groen-landicum Oeder Vaccinium vitis-idaea L Vaccinium u1iginosum Land Empetrum nigrum L and a thick nearly continuous moss layerof H splendens Aulacomnium palustre (Hedw) Schwaegr Pleuto-zium schreberi (Brid) Mitt and Sphagnum L species

23 Soils

Soils of the five successional stages are alluvial and have coarse-loamy texture but each successional stage differs in soil classifi-cation (Ping and Johnson 2000) Willow soils are classified as TypicCryofluvents alder soils as Typic Cryaquents balsam poplar soils asFluvaquentic Cryaquepts white spruce soils as Aquic Eutrocryeptsand black spruce soils as Typic Historthels All soils from the TananaRiver floodplain are generally cold wet and poorly developedRoots tend to be concentrated close to the soil surface with nearly90 of annual fine root production located in the top 30 cm of mid-successional soils (Riless et al 2006) Organic matter accumulatesover succession with forest floor thickness ranging from 0 cm inwillow to 30 cm in black spruce soils (Table1 Viereck et al 1993a)Soil temperature generally decreases over succession and

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permafrost occurs in the late successional stages of white and blackspruce (Table 1 Viereck et al 1993a)

24 Soil sampling and extraction

We sampled soils three times during 2001 June just after soilthaw early August in the middle of the growing season and lateSeptember just prior to freeze-up For each sampling event weused a stainless steel corer to obtain eight soil cores (58 cmdiameter x 20 cm depth) starting at the top of the Oe horizon (afterclearing off the fresh Oi litter horizon) along a randomized coor-dinate grid system in each 900 m2 replicate plot of the fivesuccessional stages Generally two plots were sampled per day fora total sampling duration of eight days The three replicate plots foreach successional stage were sampled successively

Numerous precautions were taken to ensure minimal effects onamino acids before soil samples were chemically extracted Afterfield coring soil cores were transported in an ice-filled cooler toa temporary field laboratory where all samples were extracted andextracts frozen within 4 h of collection In addition to minimizepotential amino acid contamination all field and laboratory equip-ment glassware and polyethylene plasticware were washed in soapand water acid-washed (10 HCI) and thoroughly rinsed in nano-pure water before use All glass fiber filters foil and glassware wereheated in a muffle furnace for 8 h at 450degC Personnel wore PVCgloves at all times to minimize contamination during processing

We separated each soil core into organic and mineral horizonswith buried organic horizons (BOHs) greater than 05 cm thicknesspooled with the forest floor (OeOa) horizon and successive mineralhorizons combined BOHs that were less than 05 cm thick werecombined with the mineral soil Woody debris and roots gt 1 mm indiameter were removed and soils were homogenized by mixingthem inside a sterile polyethylene bag We extracted amino acids fromthe soil by gently shaking 10 g field-moist soil in 75 ml nanopurewater for 10 min in a polyethylene cup Soil extracts were vacuum-filtered through a Whatman GFA glass fiber filter and an aliquot ofsupernatant was filtered by syringe through a Whatman 02 um GDXpolyvinylidene fluoride (PVDF) membrane filter into sterile 20 mlcryovials We used PVDF membrane filters because they are hydro-philic and do not bind proteins and the 02 um pore size eliminatedthe majority of microbes that could alter the amino acid pool byutilization or lysis Immediately after filtration soil extracts werefrozen and stored in a freezer until analysis Three procedural blanksof nanopure water were extracted for each replicate plot

We determined soil moisture content by oven drying a subsampleof each soil (60 oC for organic soils and 105oC for mineral soils) until

constant mass (48 h) We measured soil pH of air-dried soil samplescollected in September on a Denver Instrument Company Model 220pHconductivity meter using soil to water ratios of 15 for organicsoils and 12 for mineral soils (Hendershot et al 1993) Oven-driedSeptember soils were ground with mortar and pestle and total soilcarbon (C) and N concentrations were analyzed on a LECO CNS-2000(LECO Corporation Stjoseph MIUSA)

25 Chemical analysis by HPLC

Concentrations of individual amino acids were determined byhigh performance liquid chromatography (HPLC Gilson Inc Mid-dleton WI USA)with an automated pre-column o-phthalaldehyde(OPA)2-mercaptoethanol derivatization procedure modified fromJarret et al (1986) The automated protocol ensured samplereproducibility consistency of OPA derivatization reaction timesand enabled 24-h HPLC operation for over one thousand samplesGood peak separation was obtained using an Adsorbosphere OPA-HS 5 um column (100 x 46 mm) with accompanying 75 x 46 mmguard column (Alltech Associates Inc Deerfield ILUSA) A FD-100filter fluorescence detector (Groton Technology IncSpectroVisionActon MA USA) with a 326 nm excitation filter 470 nm emissionfilter and pulsed xenon lamp was used to detect amino acid fluo-rescence after column separation

We identified eighteen of the twenty primary i-amino acids theexceptions were proline (no reaction with OPA derivative Roth1971) and cysteine (yields very low fluorescence Roth 1971 Jarretet al 1986) All amino acid peaks eluted within 35 min The mergedpeaks of glutamic acidasparagine and histidineglutamine wereseparated and all eighteen individual amino acid peak areas wereintegrated using PeakFit software (Version 411 SYSTAT Softwarelnc Point Richmond CAUSA) with manual baseline subtractionSavitsky-Golay smoothing and an Exponentially Modified Gaussianpeak fitting model (SYSTATSoftware lnc 2002) Amino acidconcentrations were calculated from peak areas using a calibrationcurve of known amino acid standards (A9656 food hydrolysateamino acid standard with addition of L-glutamine and L-asparaginestandards Sigma-Aldrich St Louis MO USA) run on the HPLCthesame day The series of amino acid standards covered the range ofmeasured sample values Any amino acid peaks detected in theprocedural blanks were subtracted from the samples

26 Calculations

Although we measured soil properties and amino acid concen-tration on both organic and mineral horizons within a soil core we

NR Werdin-Pjisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1213

present results of soil properties and amino acid contents with theorganic and mineral horizons combined (ie the complete 0-20 cmdepth soil corer Combining the organic and mineral horizonsallowed for effective comparisons across the successional sequencewhere organic horizons made a progressively greater fraction of thetotal core volume over succession ranging from 0 for willow to79 for black spruce To calculate the soil amino acid concentrationof the combined organic and mineral horizons we multiplied theindividual horizon concentration by the horizon percent massfraction of the entire 20 cm soil then added the two horizoncomponents together The final concentration unit is ng N g - 1 drysoil where the g-l dry soil represents 1 g of the entire 20 cm core

Total free amino acid (TFAA) concentration was calculated fromthe sum of all eighteen amino acids The composition of the aminoacid pool is expressed as a proportion of the total by dividing eachindividual amino acid concentration by the TFAA concentrationProportions thereby standardized a wide range of concentrationsand allowed for effective comparisons of amino acid compositionamong the different successional stages We also calculated soilamino acid concentration on an areal basis by multiplying theconcentration (ng N g-1 dry soil) by the soil bulk density anddepth of soil core The areal unit is useful because bulk density ofthe 0-20 cm soil depth declines substantially across the succes-sional sequence

27 Statistical analysis

Successional and seasonal differences in amino acid composi-tion and concentration were tested with the UNIVARIATE CORRand MIXED procedures of SAS version 82 (SAS Institute Inc CaryNC USA) We examined differences across the successionalsequence using a nested repeated measures mixed analysis withall months combined We examined differences among the monthsover the growing season using a nested repeated measures mixedanalysis with each successional stage analyzed separately Nearlyall of the soil amino acid data were transformed (square root log orarcsine square root) to meet the assumptions of normality andhomoscedasticity We used the autoregressive covariance structurefor repeated measures analyses (Littell et al 2002) and Tukey orTukey-Kramer adjustments to post hoc significance testsComparisons were deemed statistically significant when P valueswere lt 005 The eight soil cores collected from each plot were usedin all statistical analyses however the three replicated plots of eachsuccessional stage represent the true experimental units Accord-ingly plot was treated as a nested and random factor in eachanalysis and plot was the repeated subject in the repeatedmeasures design The number of stand level replicates was threewhen each successional stage or each month was analyzed sepa-rately and nine when all months were combined over the growingseason Values presented in tables and figures are the means plusmn 1SEM of the untransformed data Relationships between soilparameters and amino acid concentration were tested withSpearmans correlations Absolute values of correlation coefficientsgt07000 with P values lt00001 were deemed as strongly corre-lated (Hatcher and Stepanski 1994)

3 Results

31 Successional patterns

311 Soil propertiesThe five successional stages showed marked differences in soil

properties (Table 2) Gravimetric moisture content was lowest inbalsam poplar and highest in black spruce Bulk density and soil pHgenerally decreased across the successional sequence Soil C and N

concentrations increased across the successional sequenceConsequently the CN ratios of soils from the coniferous stages ofwhite and black spruce were higher than the CN ratio of the soilsfrom the first three deciduous stages Soil C pools increasedprogressively over the successional sequence whereas soil N poolswere lowest in willow peaked in balsam poplar and remainedessentially unchanged from balsam poplar to black spruce

312 Amino acid compositionSoil amino acid composition was diverse with eighteen of the

twenty primary amino acids detected in most plots (Table 3)Despite the diversity of amino acids found the composition of theamino acid pool was very similar with six amino acids dominant inall successional stages alanine asparagine aspartic acid glutamicacid glutamine and histidine These six amino acids accounted for71-86 of the TFAA concentration Across the successionalsequence proportions of the six dominant amino acids ranged from17-24 for glutamic acid 11-26 for glutamine 9-15 for alanine9-14 for aspartic acid 7-13 for asparagine and 3-11 for histi-dine In all successional stages there were low proportions (0-3)of isoleucine leucine lysine methionine phenylalanine trypto-phan and tyrosine

The six amino acids prevalent across all successional stages(alanine asparagine aspartic acid glutamic acid glutamine andhistidine) include a variety of molecular sizes CN ratios chargesand solubilities Five of the six dominant amino acids are polar withalanine (non-polar) as the exception Polar amino acids averaged77 of the total amino acid pool for all successional stages Non-polar amino acids and those containing aromatic rings (phenylal-anine tryptophan and tyrosine) comprised a low proportion of theamino acid pool

Despite the similar overall composition of the amino acid poolacross the successional sequence there were several notabledifferences (Fig 1 a) Proportions of glutamine were significantlyhigher in the willow and black spruce stages tban in the balsampoplar and white spruce stages Histidine increased in proportionacross the successional sequence and the deciduous (willow alderand balsam poplar) stages had significantly lower histidineproportions than the coniferous (white and black spruce) succes-sional stages (P lt 0001) Finally alder had lower proportions ofalanine and aspartic acid than the other successional stages butalder had a higher proportion (7) of arginine

313 Amino acid concentrationWhereas the overall composition of the amino acid pool was

relatively similar among successional stages concentrations of soilamino acids were extremely different across the successionalsequence (Fig 1b Table 3) Individual amino acid concentrationsgenerally increased by an order of magnitude across the succes-sional sequence and TFAA concentration ranged from 438 ng N g-I

dry soil for willow to 4867 ng N g-1 dry soil for black spruce Therewere highly significant differences (P lt 0001) among the succes-sional stages for each of the six dominant amino acids and TFAAconcentrations Moreover the deciduous stages had significantlylower (P lt 0001) amino acid concentrations than the coniferoussuccessional stages Each of the six dominant amino acids and TFAAconcentrations was negatively correlated with soil pH and bulkdensity but positively correlated with soil C and N concentrations(P lt 0001 for all) which shows the influence of increasing organicmatter across succession

Patterns of soil amino acid concentration across the successionalsequence changed dramatically when calculated on a soil arealbasis (Fig 1c) The high amino acid concentrations in black sprucewere diminished by the lower bulk density in black spruce soilsNevertheless black spruce still had the highest glutamine glutamic

1214 NR Werden-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220

acid histidine and TFAA concentrations on an areal basis across thesuccessional sequence TFAA concentration increased acrosssuccession ranging from 70 mg N m-2 for willow to 104 mg N m-2

for black spruce and TFAA concentration was 15 times greater inblack spruce than in willow Glutamine concentrations weresignificantly greater in black spruce than in alder balsam poplarand white spruce Glutamic acid concentrations were significantlylower in the shrub successional stages than in the tree stages(Plt 0001) Histidine concentrations increased across successionwhen calculated on a soil areal basis similar to the pattern ofincreasing histidine concentration across succession that wascalculated on a soil mass basis (Fig 1b)

32 Seasonal dynamics

321 Amino acid compositionComposition of the soil amino acid pool was fairly constant

across the growing season for each successional stage (Fig 2) Theonly trend was that proportions of glutamine were highest inSeptember for all successional stages Pairwise comparisonsshowed that glutamine proportions were significantly higher(Plt 005) in September than in August for the alder balsam poplarand white spruce stages

322 Amino acid concentrationConcentrations of soil amino acids in each successional stage

showed few clear and consistent patterns during the growingseason (Fig 3) Glutamic acid concentrations tended to be highestin June for the four earliest successional stages but the differenceswere not statistically significant The balsam poplar stage exhibited

the greatest variation in seasonal patterns of amino acid concen-trations In this successional stage twelve of the eighteen aminoacids (including the six dominant) had the highest concentrationsin June although these concentrations were not always statisticallysignificant Despite predictable variations in soil temperature andmoisture concentrations of TFAA showed no clear seasonalpatterns in any successional stage (Fig 4)

4 Discussion

41 Soil amino acid composition across succession

Despite major differences in terrace age vegetation inputsproductivity and soil properties the composition of the amino acidpool was remarkably similar in each of the five successional stagesAmino acid production and consumption in these soils entaila variety of sources (eg overstory and understory plant litter rootexudates animals and microbial cells) and processes (eg plantuptake microbial uptake and abiotic sorption) In addition theproduction and utilization of amino acids within plant or microbialcells as well as the ecosystem processes linking them likely sharethe same biochemistry and amino acid constituents regardless ofvegetation type soil environment or climate Although there arethese commonalities the capacity of plants and microbes to utilizeamino acids has been shown to vary among different plant speciesand functional types (Chapin et al 1993 Kielland 1994 Weigeltet al 2005) with amino acid molecular size (Kielland1994) aswell as being correlated with the concentration of the amino acidpool (Kielland 1994 Jones et al 2005a)

Elucidating the potential origin of amino acids in the soil iscomplicated due to the intertwined nature and speed of rhizo-sphere dynamics Analysis of amino acid composition of overstoryplant litter in these successional stages shows high proportions ofalanine glutamine and histidine similar to soil (unpublishedobservations) Other amino acids commonly found in arctic plantsinclude alanine arginine asparagine glutamine and glutamic acid(Chapin et aI 1986) the amino acid pool in boreal forest vegetation(shrub herb and grass foliage) is dominated by asparagine gluta-mine and arginine (Ohlson et al 1995) In addition to the aminoacids found in plant litter asparagine and glutamine are commonconstituents of plant xylem and phloem (Pate 1973 Kielland 1994Rochat 2001) whereas asparagine glutamine and arginine arecommon storage amino acids in above and belowground planttissue (Chapin et al 1986 Nasholm et al 1994 Ohlson et al 1995Nordin and Nasholm 1997) Microorganisms whose cell walls havehigh proportions of alanine aspartic acid and glutamic acid (Frie-del and Scheller 2002 Yu et al 2002) are also a likely source ofamino acids in the soil

A review of other studies demonstrates that there are commonsoil amino acids across a variety of natural and managed ecosys-tems from different climates further supporting the view that soilamino acids originate from similar ecosystem components orthrough similar biochemical processes (Table 4) Alanine and

NR Werdin-Pfisterer et a1 Soil Biology amp Biochemistry 41 (2009) 1210-1220 1215

glutamic acid are two common important amino acids acrossa variety of ecosystems Notably missing from our results were thelarge proportions of the amino acids arginine glycine and serinefound in nearly every other study including those in the borealforest in Sweden (Nordin et al 2001) and Alaskan tundra (Kielland1995) We did detect a large proportion (7) of arginine in the aldersuccessional stage perhaps because alder transports amino acidsfrom their N-fixing root nodules in the form of citrulline (Pate1973 Gaudillere 2001) a precursor to arginine In addition argi-nine is an efficient N storage compound (Chapin et al 1986) Nordinand Nasholm 1997) facilitating N storage during rapid N-fixation(Uliassi and Ruess 2002) Only a few studies report data forasparagine glutamine and histidine but these amino acids havebeen found to be important components of the amino acid pool(Ivarson and Sowden 1969 Abuarghub and Read 1988) Nordinet al 2001 Yu et al 2002) The amides asparagine and glutaminecombined made up 27 of the soil amino acid pool in both theboreal forest in Sweden (Nordin et al 2001) and the Alaskan borealforest of this study Only three other studies found similarly largeproportions of aspartic acid (Ivarson and Sowden 1969 Read and

Bajwa 1985 Kielland 1995) Several amino acids were found in lowproportions across all studies isoleucine methionine phenylala-nine tryptophan and tyrosine The similar amino acid compositionacross a variety of different soils is consistent with reviews byAbuarghub and Read (1988) and Schulten and Schnitzer (1998)

There are only a handful of studies describing the amino acidcomposition in soil but the majority of laboratory and field studiesof amino acid uptake have focused on glycine Results of thisstudy indicate that glycine accounted for only 3 of the TFAA pool(Table 4) Thus we suggest that the influence of specific aminoacids to plant nutrition would be strengthened by further exami-nation of the abundance of these individual amino acids in the soil

42 Soil amino acid concentration cross succession

Amino acid concentrations generally increased across thesuccessional sequence (Fig 1b and c) probably due simply to theaccumulation of soil organic matter (Tables 1 and 2 Viereck et al1993a) which is a source of amino acids In addition boreal forestsoils high in organic matter also have high fungal biomass

1216 NR Werdin-Pfisterer et at Soil Biology amp Biochemistry 41 (2009) 1210-1220

(F1anafan and Van Cleve 1983) which is another source of aminoacids Kielland et al (2007) found that soil protease activityincreased across succession on the same plots sampled in this studyand suggested that the high concentrations of soil amino acidsin the later successional stages were sustained through highproteolytic activity

As organic matter accumulates across succession there is anassociated increase in cation exchange capacity (Van Cleve et al1993a) so more amino acids may be adsorbed onto the soilexchange sites of the later successional stages The basic aminoacid histidine should develop a strong positive charge in the lowpH conditions of late successional soils so the increase in histi-dine proportion and concentration over succession (Fig 1a-c)may reflect strong adsorption onto a greater number of soilexchange sites in the later successional stages Howeverproportions and concentrations of the other basic amino acidsarginine and lysine did not consistently increase across succes-sion (Table 3) so an explanation for the increased histidine

proportion and concentration remains uncertain A similarobservation was reported for heathland soils (Abuarghub andRead 1988)

Another possible explanation for the increased concentration ofsoil amino acids across succession is that the later successionalstages have greater belowground allocation in the form of fine roots(Ruess et al 2006) In the boreal forest belowground inputs fromroot turnover are greater and decompose faster than the above-ground litter inputs (Ruess et al 1996) The large spike in propor-tion and concentration of glutamine in the black sprucesuccessional stage (Fig 1 a and b) may result from greater quantitiesof root exudation or lysis from this active root biomass The patternof glutamine proportion and glutamine concentration per unit areaacross succession (Fig1 a and c) mirrors the pattern of below-ground fine root production across succession that Ruess et al(2006) measured using minirhizotrons We found high proportionsand concentrations of glutamine in the willow and black sprucestages where Ruess et al (2006) found the greatest allocation of

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1217

belowground production whereas we found a low proportion andconcentration of glutamine in the balsam poplar stage that had thelowest belowground allocation

43 Seasonal dynamics of soil amino acids

The composition and concentration of the soil amino acid poolappeared relatively constant over the growing season for eachsuccessional stage in contrast to our original hypothesis anddespite major seasonal changes in plant productivity litterfallmicrobial freeze-thaw flushes and soil temperatures Most studieshave found that amino acid concentrations do vary significantlyacross the season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005 though seeNordin et al 2001 Jones et al 2005a) Detecting seasonal changesif present may be problematic given that soil amino acids canfluctuate from high to low concentrations in less than a week(Weintraub and Schimel 2005) We sampled soils over three

months at monthly intervals which may constitute a rather coarsetemporal resolution

44 TFAA concentration compared to other studies

The range of TFAA concentrations of this study is within therange of values reported for a variety of ecosystems and climatesTable 4) Our mean TFAA values (20-350 nmol amino acid g 1 drysoil 04-54 ug amino acid N g 1 dry soil or 40-300 um) are similaror slightly higher than soil TFAA concentrations in the boreal forestin Sweden (5-455 nmol amino acid g 1 dry soil Persson and Nasholm 2001) the alpine dry meadows of Colorado (03-30 ug amino acid Ng-l dry soil Lipson et al 1999) and the mixedconifer and pygmy forests in northern California (16-299 um Yuet al 2002) We caution however about making inferences fromthese comparisons due to differences in soil sampling depthorganic matter content and extraction methods among the studies(Jones and Willett 2006)

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

1212 NR Werdill-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220

permafrost occurs in the late successional stages of white and blackspruce (Table 1 Viereck et al 1993a)

24 Soil sampling and extraction

We sampled soils three times during 2001 June just after soilthaw early August in the middle of the growing season and lateSeptember just prior to freeze-up For each sampling event weused a stainless steel corer to obtain eight soil cores (58 cmdiameter x 20 cm depth) starting at the top of the Oe horizon (afterclearing off the fresh Oi litter horizon) along a randomized coor-dinate grid system in each 900 m2 replicate plot of the fivesuccessional stages Generally two plots were sampled per day fora total sampling duration of eight days The three replicate plots foreach successional stage were sampled successively

Numerous precautions were taken to ensure minimal effects onamino acids before soil samples were chemically extracted Afterfield coring soil cores were transported in an ice-filled cooler toa temporary field laboratory where all samples were extracted andextracts frozen within 4 h of collection In addition to minimizepotential amino acid contamination all field and laboratory equip-ment glassware and polyethylene plasticware were washed in soapand water acid-washed (10 HCI) and thoroughly rinsed in nano-pure water before use All glass fiber filters foil and glassware wereheated in a muffle furnace for 8 h at 450degC Personnel wore PVCgloves at all times to minimize contamination during processing

We separated each soil core into organic and mineral horizonswith buried organic horizons (BOHs) greater than 05 cm thicknesspooled with the forest floor (OeOa) horizon and successive mineralhorizons combined BOHs that were less than 05 cm thick werecombined with the mineral soil Woody debris and roots gt 1 mm indiameter were removed and soils were homogenized by mixingthem inside a sterile polyethylene bag We extracted amino acids fromthe soil by gently shaking 10 g field-moist soil in 75 ml nanopurewater for 10 min in a polyethylene cup Soil extracts were vacuum-filtered through a Whatman GFA glass fiber filter and an aliquot ofsupernatant was filtered by syringe through a Whatman 02 um GDXpolyvinylidene fluoride (PVDF) membrane filter into sterile 20 mlcryovials We used PVDF membrane filters because they are hydro-philic and do not bind proteins and the 02 um pore size eliminatedthe majority of microbes that could alter the amino acid pool byutilization or lysis Immediately after filtration soil extracts werefrozen and stored in a freezer until analysis Three procedural blanksof nanopure water were extracted for each replicate plot

We determined soil moisture content by oven drying a subsampleof each soil (60 oC for organic soils and 105oC for mineral soils) until

constant mass (48 h) We measured soil pH of air-dried soil samplescollected in September on a Denver Instrument Company Model 220pHconductivity meter using soil to water ratios of 15 for organicsoils and 12 for mineral soils (Hendershot et al 1993) Oven-driedSeptember soils were ground with mortar and pestle and total soilcarbon (C) and N concentrations were analyzed on a LECO CNS-2000(LECO Corporation Stjoseph MIUSA)

25 Chemical analysis by HPLC

Concentrations of individual amino acids were determined byhigh performance liquid chromatography (HPLC Gilson Inc Mid-dleton WI USA)with an automated pre-column o-phthalaldehyde(OPA)2-mercaptoethanol derivatization procedure modified fromJarret et al (1986) The automated protocol ensured samplereproducibility consistency of OPA derivatization reaction timesand enabled 24-h HPLC operation for over one thousand samplesGood peak separation was obtained using an Adsorbosphere OPA-HS 5 um column (100 x 46 mm) with accompanying 75 x 46 mmguard column (Alltech Associates Inc Deerfield ILUSA) A FD-100filter fluorescence detector (Groton Technology IncSpectroVisionActon MA USA) with a 326 nm excitation filter 470 nm emissionfilter and pulsed xenon lamp was used to detect amino acid fluo-rescence after column separation

We identified eighteen of the twenty primary i-amino acids theexceptions were proline (no reaction with OPA derivative Roth1971) and cysteine (yields very low fluorescence Roth 1971 Jarretet al 1986) All amino acid peaks eluted within 35 min The mergedpeaks of glutamic acidasparagine and histidineglutamine wereseparated and all eighteen individual amino acid peak areas wereintegrated using PeakFit software (Version 411 SYSTAT Softwarelnc Point Richmond CAUSA) with manual baseline subtractionSavitsky-Golay smoothing and an Exponentially Modified Gaussianpeak fitting model (SYSTATSoftware lnc 2002) Amino acidconcentrations were calculated from peak areas using a calibrationcurve of known amino acid standards (A9656 food hydrolysateamino acid standard with addition of L-glutamine and L-asparaginestandards Sigma-Aldrich St Louis MO USA) run on the HPLCthesame day The series of amino acid standards covered the range ofmeasured sample values Any amino acid peaks detected in theprocedural blanks were subtracted from the samples

26 Calculations

Although we measured soil properties and amino acid concen-tration on both organic and mineral horizons within a soil core we

NR Werdin-Pjisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1213

present results of soil properties and amino acid contents with theorganic and mineral horizons combined (ie the complete 0-20 cmdepth soil corer Combining the organic and mineral horizonsallowed for effective comparisons across the successional sequencewhere organic horizons made a progressively greater fraction of thetotal core volume over succession ranging from 0 for willow to79 for black spruce To calculate the soil amino acid concentrationof the combined organic and mineral horizons we multiplied theindividual horizon concentration by the horizon percent massfraction of the entire 20 cm soil then added the two horizoncomponents together The final concentration unit is ng N g - 1 drysoil where the g-l dry soil represents 1 g of the entire 20 cm core

Total free amino acid (TFAA) concentration was calculated fromthe sum of all eighteen amino acids The composition of the aminoacid pool is expressed as a proportion of the total by dividing eachindividual amino acid concentration by the TFAA concentrationProportions thereby standardized a wide range of concentrationsand allowed for effective comparisons of amino acid compositionamong the different successional stages We also calculated soilamino acid concentration on an areal basis by multiplying theconcentration (ng N g-1 dry soil) by the soil bulk density anddepth of soil core The areal unit is useful because bulk density ofthe 0-20 cm soil depth declines substantially across the succes-sional sequence

27 Statistical analysis

Successional and seasonal differences in amino acid composi-tion and concentration were tested with the UNIVARIATE CORRand MIXED procedures of SAS version 82 (SAS Institute Inc CaryNC USA) We examined differences across the successionalsequence using a nested repeated measures mixed analysis withall months combined We examined differences among the monthsover the growing season using a nested repeated measures mixedanalysis with each successional stage analyzed separately Nearlyall of the soil amino acid data were transformed (square root log orarcsine square root) to meet the assumptions of normality andhomoscedasticity We used the autoregressive covariance structurefor repeated measures analyses (Littell et al 2002) and Tukey orTukey-Kramer adjustments to post hoc significance testsComparisons were deemed statistically significant when P valueswere lt 005 The eight soil cores collected from each plot were usedin all statistical analyses however the three replicated plots of eachsuccessional stage represent the true experimental units Accord-ingly plot was treated as a nested and random factor in eachanalysis and plot was the repeated subject in the repeatedmeasures design The number of stand level replicates was threewhen each successional stage or each month was analyzed sepa-rately and nine when all months were combined over the growingseason Values presented in tables and figures are the means plusmn 1SEM of the untransformed data Relationships between soilparameters and amino acid concentration were tested withSpearmans correlations Absolute values of correlation coefficientsgt07000 with P values lt00001 were deemed as strongly corre-lated (Hatcher and Stepanski 1994)

3 Results

31 Successional patterns

311 Soil propertiesThe five successional stages showed marked differences in soil

properties (Table 2) Gravimetric moisture content was lowest inbalsam poplar and highest in black spruce Bulk density and soil pHgenerally decreased across the successional sequence Soil C and N

concentrations increased across the successional sequenceConsequently the CN ratios of soils from the coniferous stages ofwhite and black spruce were higher than the CN ratio of the soilsfrom the first three deciduous stages Soil C pools increasedprogressively over the successional sequence whereas soil N poolswere lowest in willow peaked in balsam poplar and remainedessentially unchanged from balsam poplar to black spruce

312 Amino acid compositionSoil amino acid composition was diverse with eighteen of the

twenty primary amino acids detected in most plots (Table 3)Despite the diversity of amino acids found the composition of theamino acid pool was very similar with six amino acids dominant inall successional stages alanine asparagine aspartic acid glutamicacid glutamine and histidine These six amino acids accounted for71-86 of the TFAA concentration Across the successionalsequence proportions of the six dominant amino acids ranged from17-24 for glutamic acid 11-26 for glutamine 9-15 for alanine9-14 for aspartic acid 7-13 for asparagine and 3-11 for histi-dine In all successional stages there were low proportions (0-3)of isoleucine leucine lysine methionine phenylalanine trypto-phan and tyrosine

The six amino acids prevalent across all successional stages(alanine asparagine aspartic acid glutamic acid glutamine andhistidine) include a variety of molecular sizes CN ratios chargesand solubilities Five of the six dominant amino acids are polar withalanine (non-polar) as the exception Polar amino acids averaged77 of the total amino acid pool for all successional stages Non-polar amino acids and those containing aromatic rings (phenylal-anine tryptophan and tyrosine) comprised a low proportion of theamino acid pool

Despite the similar overall composition of the amino acid poolacross the successional sequence there were several notabledifferences (Fig 1 a) Proportions of glutamine were significantlyhigher in the willow and black spruce stages tban in the balsampoplar and white spruce stages Histidine increased in proportionacross the successional sequence and the deciduous (willow alderand balsam poplar) stages had significantly lower histidineproportions than the coniferous (white and black spruce) succes-sional stages (P lt 0001) Finally alder had lower proportions ofalanine and aspartic acid than the other successional stages butalder had a higher proportion (7) of arginine

313 Amino acid concentrationWhereas the overall composition of the amino acid pool was

relatively similar among successional stages concentrations of soilamino acids were extremely different across the successionalsequence (Fig 1b Table 3) Individual amino acid concentrationsgenerally increased by an order of magnitude across the succes-sional sequence and TFAA concentration ranged from 438 ng N g-I

dry soil for willow to 4867 ng N g-1 dry soil for black spruce Therewere highly significant differences (P lt 0001) among the succes-sional stages for each of the six dominant amino acids and TFAAconcentrations Moreover the deciduous stages had significantlylower (P lt 0001) amino acid concentrations than the coniferoussuccessional stages Each of the six dominant amino acids and TFAAconcentrations was negatively correlated with soil pH and bulkdensity but positively correlated with soil C and N concentrations(P lt 0001 for all) which shows the influence of increasing organicmatter across succession

Patterns of soil amino acid concentration across the successionalsequence changed dramatically when calculated on a soil arealbasis (Fig 1c) The high amino acid concentrations in black sprucewere diminished by the lower bulk density in black spruce soilsNevertheless black spruce still had the highest glutamine glutamic

1214 NR Werden-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220

acid histidine and TFAA concentrations on an areal basis across thesuccessional sequence TFAA concentration increased acrosssuccession ranging from 70 mg N m-2 for willow to 104 mg N m-2

for black spruce and TFAA concentration was 15 times greater inblack spruce than in willow Glutamine concentrations weresignificantly greater in black spruce than in alder balsam poplarand white spruce Glutamic acid concentrations were significantlylower in the shrub successional stages than in the tree stages(Plt 0001) Histidine concentrations increased across successionwhen calculated on a soil areal basis similar to the pattern ofincreasing histidine concentration across succession that wascalculated on a soil mass basis (Fig 1b)

32 Seasonal dynamics

321 Amino acid compositionComposition of the soil amino acid pool was fairly constant

across the growing season for each successional stage (Fig 2) Theonly trend was that proportions of glutamine were highest inSeptember for all successional stages Pairwise comparisonsshowed that glutamine proportions were significantly higher(Plt 005) in September than in August for the alder balsam poplarand white spruce stages

322 Amino acid concentrationConcentrations of soil amino acids in each successional stage

showed few clear and consistent patterns during the growingseason (Fig 3) Glutamic acid concentrations tended to be highestin June for the four earliest successional stages but the differenceswere not statistically significant The balsam poplar stage exhibited

the greatest variation in seasonal patterns of amino acid concen-trations In this successional stage twelve of the eighteen aminoacids (including the six dominant) had the highest concentrationsin June although these concentrations were not always statisticallysignificant Despite predictable variations in soil temperature andmoisture concentrations of TFAA showed no clear seasonalpatterns in any successional stage (Fig 4)

4 Discussion

41 Soil amino acid composition across succession

Despite major differences in terrace age vegetation inputsproductivity and soil properties the composition of the amino acidpool was remarkably similar in each of the five successional stagesAmino acid production and consumption in these soils entaila variety of sources (eg overstory and understory plant litter rootexudates animals and microbial cells) and processes (eg plantuptake microbial uptake and abiotic sorption) In addition theproduction and utilization of amino acids within plant or microbialcells as well as the ecosystem processes linking them likely sharethe same biochemistry and amino acid constituents regardless ofvegetation type soil environment or climate Although there arethese commonalities the capacity of plants and microbes to utilizeamino acids has been shown to vary among different plant speciesand functional types (Chapin et al 1993 Kielland 1994 Weigeltet al 2005) with amino acid molecular size (Kielland1994) aswell as being correlated with the concentration of the amino acidpool (Kielland 1994 Jones et al 2005a)

Elucidating the potential origin of amino acids in the soil iscomplicated due to the intertwined nature and speed of rhizo-sphere dynamics Analysis of amino acid composition of overstoryplant litter in these successional stages shows high proportions ofalanine glutamine and histidine similar to soil (unpublishedobservations) Other amino acids commonly found in arctic plantsinclude alanine arginine asparagine glutamine and glutamic acid(Chapin et aI 1986) the amino acid pool in boreal forest vegetation(shrub herb and grass foliage) is dominated by asparagine gluta-mine and arginine (Ohlson et al 1995) In addition to the aminoacids found in plant litter asparagine and glutamine are commonconstituents of plant xylem and phloem (Pate 1973 Kielland 1994Rochat 2001) whereas asparagine glutamine and arginine arecommon storage amino acids in above and belowground planttissue (Chapin et al 1986 Nasholm et al 1994 Ohlson et al 1995Nordin and Nasholm 1997) Microorganisms whose cell walls havehigh proportions of alanine aspartic acid and glutamic acid (Frie-del and Scheller 2002 Yu et al 2002) are also a likely source ofamino acids in the soil

A review of other studies demonstrates that there are commonsoil amino acids across a variety of natural and managed ecosys-tems from different climates further supporting the view that soilamino acids originate from similar ecosystem components orthrough similar biochemical processes (Table 4) Alanine and

NR Werdin-Pfisterer et a1 Soil Biology amp Biochemistry 41 (2009) 1210-1220 1215

glutamic acid are two common important amino acids acrossa variety of ecosystems Notably missing from our results were thelarge proportions of the amino acids arginine glycine and serinefound in nearly every other study including those in the borealforest in Sweden (Nordin et al 2001) and Alaskan tundra (Kielland1995) We did detect a large proportion (7) of arginine in the aldersuccessional stage perhaps because alder transports amino acidsfrom their N-fixing root nodules in the form of citrulline (Pate1973 Gaudillere 2001) a precursor to arginine In addition argi-nine is an efficient N storage compound (Chapin et al 1986) Nordinand Nasholm 1997) facilitating N storage during rapid N-fixation(Uliassi and Ruess 2002) Only a few studies report data forasparagine glutamine and histidine but these amino acids havebeen found to be important components of the amino acid pool(Ivarson and Sowden 1969 Abuarghub and Read 1988) Nordinet al 2001 Yu et al 2002) The amides asparagine and glutaminecombined made up 27 of the soil amino acid pool in both theboreal forest in Sweden (Nordin et al 2001) and the Alaskan borealforest of this study Only three other studies found similarly largeproportions of aspartic acid (Ivarson and Sowden 1969 Read and

Bajwa 1985 Kielland 1995) Several amino acids were found in lowproportions across all studies isoleucine methionine phenylala-nine tryptophan and tyrosine The similar amino acid compositionacross a variety of different soils is consistent with reviews byAbuarghub and Read (1988) and Schulten and Schnitzer (1998)

There are only a handful of studies describing the amino acidcomposition in soil but the majority of laboratory and field studiesof amino acid uptake have focused on glycine Results of thisstudy indicate that glycine accounted for only 3 of the TFAA pool(Table 4) Thus we suggest that the influence of specific aminoacids to plant nutrition would be strengthened by further exami-nation of the abundance of these individual amino acids in the soil

42 Soil amino acid concentration cross succession

Amino acid concentrations generally increased across thesuccessional sequence (Fig 1b and c) probably due simply to theaccumulation of soil organic matter (Tables 1 and 2 Viereck et al1993a) which is a source of amino acids In addition boreal forestsoils high in organic matter also have high fungal biomass

1216 NR Werdin-Pfisterer et at Soil Biology amp Biochemistry 41 (2009) 1210-1220

(F1anafan and Van Cleve 1983) which is another source of aminoacids Kielland et al (2007) found that soil protease activityincreased across succession on the same plots sampled in this studyand suggested that the high concentrations of soil amino acidsin the later successional stages were sustained through highproteolytic activity

As organic matter accumulates across succession there is anassociated increase in cation exchange capacity (Van Cleve et al1993a) so more amino acids may be adsorbed onto the soilexchange sites of the later successional stages The basic aminoacid histidine should develop a strong positive charge in the lowpH conditions of late successional soils so the increase in histi-dine proportion and concentration over succession (Fig 1a-c)may reflect strong adsorption onto a greater number of soilexchange sites in the later successional stages Howeverproportions and concentrations of the other basic amino acidsarginine and lysine did not consistently increase across succes-sion (Table 3) so an explanation for the increased histidine

proportion and concentration remains uncertain A similarobservation was reported for heathland soils (Abuarghub andRead 1988)

Another possible explanation for the increased concentration ofsoil amino acids across succession is that the later successionalstages have greater belowground allocation in the form of fine roots(Ruess et al 2006) In the boreal forest belowground inputs fromroot turnover are greater and decompose faster than the above-ground litter inputs (Ruess et al 1996) The large spike in propor-tion and concentration of glutamine in the black sprucesuccessional stage (Fig 1 a and b) may result from greater quantitiesof root exudation or lysis from this active root biomass The patternof glutamine proportion and glutamine concentration per unit areaacross succession (Fig1 a and c) mirrors the pattern of below-ground fine root production across succession that Ruess et al(2006) measured using minirhizotrons We found high proportionsand concentrations of glutamine in the willow and black sprucestages where Ruess et al (2006) found the greatest allocation of

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1217

belowground production whereas we found a low proportion andconcentration of glutamine in the balsam poplar stage that had thelowest belowground allocation

43 Seasonal dynamics of soil amino acids

The composition and concentration of the soil amino acid poolappeared relatively constant over the growing season for eachsuccessional stage in contrast to our original hypothesis anddespite major seasonal changes in plant productivity litterfallmicrobial freeze-thaw flushes and soil temperatures Most studieshave found that amino acid concentrations do vary significantlyacross the season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005 though seeNordin et al 2001 Jones et al 2005a) Detecting seasonal changesif present may be problematic given that soil amino acids canfluctuate from high to low concentrations in less than a week(Weintraub and Schimel 2005) We sampled soils over three

months at monthly intervals which may constitute a rather coarsetemporal resolution

44 TFAA concentration compared to other studies

The range of TFAA concentrations of this study is within therange of values reported for a variety of ecosystems and climatesTable 4) Our mean TFAA values (20-350 nmol amino acid g 1 drysoil 04-54 ug amino acid N g 1 dry soil or 40-300 um) are similaror slightly higher than soil TFAA concentrations in the boreal forestin Sweden (5-455 nmol amino acid g 1 dry soil Persson and Nasholm 2001) the alpine dry meadows of Colorado (03-30 ug amino acid Ng-l dry soil Lipson et al 1999) and the mixedconifer and pygmy forests in northern California (16-299 um Yuet al 2002) We caution however about making inferences fromthese comparisons due to differences in soil sampling depthorganic matter content and extraction methods among the studies(Jones and Willett 2006)

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

NR Werdin-Pjisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1213

present results of soil properties and amino acid contents with theorganic and mineral horizons combined (ie the complete 0-20 cmdepth soil corer Combining the organic and mineral horizonsallowed for effective comparisons across the successional sequencewhere organic horizons made a progressively greater fraction of thetotal core volume over succession ranging from 0 for willow to79 for black spruce To calculate the soil amino acid concentrationof the combined organic and mineral horizons we multiplied theindividual horizon concentration by the horizon percent massfraction of the entire 20 cm soil then added the two horizoncomponents together The final concentration unit is ng N g - 1 drysoil where the g-l dry soil represents 1 g of the entire 20 cm core

Total free amino acid (TFAA) concentration was calculated fromthe sum of all eighteen amino acids The composition of the aminoacid pool is expressed as a proportion of the total by dividing eachindividual amino acid concentration by the TFAA concentrationProportions thereby standardized a wide range of concentrationsand allowed for effective comparisons of amino acid compositionamong the different successional stages We also calculated soilamino acid concentration on an areal basis by multiplying theconcentration (ng N g-1 dry soil) by the soil bulk density anddepth of soil core The areal unit is useful because bulk density ofthe 0-20 cm soil depth declines substantially across the succes-sional sequence

27 Statistical analysis

Successional and seasonal differences in amino acid composi-tion and concentration were tested with the UNIVARIATE CORRand MIXED procedures of SAS version 82 (SAS Institute Inc CaryNC USA) We examined differences across the successionalsequence using a nested repeated measures mixed analysis withall months combined We examined differences among the monthsover the growing season using a nested repeated measures mixedanalysis with each successional stage analyzed separately Nearlyall of the soil amino acid data were transformed (square root log orarcsine square root) to meet the assumptions of normality andhomoscedasticity We used the autoregressive covariance structurefor repeated measures analyses (Littell et al 2002) and Tukey orTukey-Kramer adjustments to post hoc significance testsComparisons were deemed statistically significant when P valueswere lt 005 The eight soil cores collected from each plot were usedin all statistical analyses however the three replicated plots of eachsuccessional stage represent the true experimental units Accord-ingly plot was treated as a nested and random factor in eachanalysis and plot was the repeated subject in the repeatedmeasures design The number of stand level replicates was threewhen each successional stage or each month was analyzed sepa-rately and nine when all months were combined over the growingseason Values presented in tables and figures are the means plusmn 1SEM of the untransformed data Relationships between soilparameters and amino acid concentration were tested withSpearmans correlations Absolute values of correlation coefficientsgt07000 with P values lt00001 were deemed as strongly corre-lated (Hatcher and Stepanski 1994)

3 Results

31 Successional patterns

311 Soil propertiesThe five successional stages showed marked differences in soil

properties (Table 2) Gravimetric moisture content was lowest inbalsam poplar and highest in black spruce Bulk density and soil pHgenerally decreased across the successional sequence Soil C and N

concentrations increased across the successional sequenceConsequently the CN ratios of soils from the coniferous stages ofwhite and black spruce were higher than the CN ratio of the soilsfrom the first three deciduous stages Soil C pools increasedprogressively over the successional sequence whereas soil N poolswere lowest in willow peaked in balsam poplar and remainedessentially unchanged from balsam poplar to black spruce

312 Amino acid compositionSoil amino acid composition was diverse with eighteen of the

twenty primary amino acids detected in most plots (Table 3)Despite the diversity of amino acids found the composition of theamino acid pool was very similar with six amino acids dominant inall successional stages alanine asparagine aspartic acid glutamicacid glutamine and histidine These six amino acids accounted for71-86 of the TFAA concentration Across the successionalsequence proportions of the six dominant amino acids ranged from17-24 for glutamic acid 11-26 for glutamine 9-15 for alanine9-14 for aspartic acid 7-13 for asparagine and 3-11 for histi-dine In all successional stages there were low proportions (0-3)of isoleucine leucine lysine methionine phenylalanine trypto-phan and tyrosine

The six amino acids prevalent across all successional stages(alanine asparagine aspartic acid glutamic acid glutamine andhistidine) include a variety of molecular sizes CN ratios chargesand solubilities Five of the six dominant amino acids are polar withalanine (non-polar) as the exception Polar amino acids averaged77 of the total amino acid pool for all successional stages Non-polar amino acids and those containing aromatic rings (phenylal-anine tryptophan and tyrosine) comprised a low proportion of theamino acid pool

Despite the similar overall composition of the amino acid poolacross the successional sequence there were several notabledifferences (Fig 1 a) Proportions of glutamine were significantlyhigher in the willow and black spruce stages tban in the balsampoplar and white spruce stages Histidine increased in proportionacross the successional sequence and the deciduous (willow alderand balsam poplar) stages had significantly lower histidineproportions than the coniferous (white and black spruce) succes-sional stages (P lt 0001) Finally alder had lower proportions ofalanine and aspartic acid than the other successional stages butalder had a higher proportion (7) of arginine

313 Amino acid concentrationWhereas the overall composition of the amino acid pool was

relatively similar among successional stages concentrations of soilamino acids were extremely different across the successionalsequence (Fig 1b Table 3) Individual amino acid concentrationsgenerally increased by an order of magnitude across the succes-sional sequence and TFAA concentration ranged from 438 ng N g-I

dry soil for willow to 4867 ng N g-1 dry soil for black spruce Therewere highly significant differences (P lt 0001) among the succes-sional stages for each of the six dominant amino acids and TFAAconcentrations Moreover the deciduous stages had significantlylower (P lt 0001) amino acid concentrations than the coniferoussuccessional stages Each of the six dominant amino acids and TFAAconcentrations was negatively correlated with soil pH and bulkdensity but positively correlated with soil C and N concentrations(P lt 0001 for all) which shows the influence of increasing organicmatter across succession

Patterns of soil amino acid concentration across the successionalsequence changed dramatically when calculated on a soil arealbasis (Fig 1c) The high amino acid concentrations in black sprucewere diminished by the lower bulk density in black spruce soilsNevertheless black spruce still had the highest glutamine glutamic

1214 NR Werden-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220

acid histidine and TFAA concentrations on an areal basis across thesuccessional sequence TFAA concentration increased acrosssuccession ranging from 70 mg N m-2 for willow to 104 mg N m-2

for black spruce and TFAA concentration was 15 times greater inblack spruce than in willow Glutamine concentrations weresignificantly greater in black spruce than in alder balsam poplarand white spruce Glutamic acid concentrations were significantlylower in the shrub successional stages than in the tree stages(Plt 0001) Histidine concentrations increased across successionwhen calculated on a soil areal basis similar to the pattern ofincreasing histidine concentration across succession that wascalculated on a soil mass basis (Fig 1b)

32 Seasonal dynamics

321 Amino acid compositionComposition of the soil amino acid pool was fairly constant

across the growing season for each successional stage (Fig 2) Theonly trend was that proportions of glutamine were highest inSeptember for all successional stages Pairwise comparisonsshowed that glutamine proportions were significantly higher(Plt 005) in September than in August for the alder balsam poplarand white spruce stages

322 Amino acid concentrationConcentrations of soil amino acids in each successional stage

showed few clear and consistent patterns during the growingseason (Fig 3) Glutamic acid concentrations tended to be highestin June for the four earliest successional stages but the differenceswere not statistically significant The balsam poplar stage exhibited

the greatest variation in seasonal patterns of amino acid concen-trations In this successional stage twelve of the eighteen aminoacids (including the six dominant) had the highest concentrationsin June although these concentrations were not always statisticallysignificant Despite predictable variations in soil temperature andmoisture concentrations of TFAA showed no clear seasonalpatterns in any successional stage (Fig 4)

4 Discussion

41 Soil amino acid composition across succession

Despite major differences in terrace age vegetation inputsproductivity and soil properties the composition of the amino acidpool was remarkably similar in each of the five successional stagesAmino acid production and consumption in these soils entaila variety of sources (eg overstory and understory plant litter rootexudates animals and microbial cells) and processes (eg plantuptake microbial uptake and abiotic sorption) In addition theproduction and utilization of amino acids within plant or microbialcells as well as the ecosystem processes linking them likely sharethe same biochemistry and amino acid constituents regardless ofvegetation type soil environment or climate Although there arethese commonalities the capacity of plants and microbes to utilizeamino acids has been shown to vary among different plant speciesand functional types (Chapin et al 1993 Kielland 1994 Weigeltet al 2005) with amino acid molecular size (Kielland1994) aswell as being correlated with the concentration of the amino acidpool (Kielland 1994 Jones et al 2005a)

Elucidating the potential origin of amino acids in the soil iscomplicated due to the intertwined nature and speed of rhizo-sphere dynamics Analysis of amino acid composition of overstoryplant litter in these successional stages shows high proportions ofalanine glutamine and histidine similar to soil (unpublishedobservations) Other amino acids commonly found in arctic plantsinclude alanine arginine asparagine glutamine and glutamic acid(Chapin et aI 1986) the amino acid pool in boreal forest vegetation(shrub herb and grass foliage) is dominated by asparagine gluta-mine and arginine (Ohlson et al 1995) In addition to the aminoacids found in plant litter asparagine and glutamine are commonconstituents of plant xylem and phloem (Pate 1973 Kielland 1994Rochat 2001) whereas asparagine glutamine and arginine arecommon storage amino acids in above and belowground planttissue (Chapin et al 1986 Nasholm et al 1994 Ohlson et al 1995Nordin and Nasholm 1997) Microorganisms whose cell walls havehigh proportions of alanine aspartic acid and glutamic acid (Frie-del and Scheller 2002 Yu et al 2002) are also a likely source ofamino acids in the soil

A review of other studies demonstrates that there are commonsoil amino acids across a variety of natural and managed ecosys-tems from different climates further supporting the view that soilamino acids originate from similar ecosystem components orthrough similar biochemical processes (Table 4) Alanine and

NR Werdin-Pfisterer et a1 Soil Biology amp Biochemistry 41 (2009) 1210-1220 1215

glutamic acid are two common important amino acids acrossa variety of ecosystems Notably missing from our results were thelarge proportions of the amino acids arginine glycine and serinefound in nearly every other study including those in the borealforest in Sweden (Nordin et al 2001) and Alaskan tundra (Kielland1995) We did detect a large proportion (7) of arginine in the aldersuccessional stage perhaps because alder transports amino acidsfrom their N-fixing root nodules in the form of citrulline (Pate1973 Gaudillere 2001) a precursor to arginine In addition argi-nine is an efficient N storage compound (Chapin et al 1986) Nordinand Nasholm 1997) facilitating N storage during rapid N-fixation(Uliassi and Ruess 2002) Only a few studies report data forasparagine glutamine and histidine but these amino acids havebeen found to be important components of the amino acid pool(Ivarson and Sowden 1969 Abuarghub and Read 1988) Nordinet al 2001 Yu et al 2002) The amides asparagine and glutaminecombined made up 27 of the soil amino acid pool in both theboreal forest in Sweden (Nordin et al 2001) and the Alaskan borealforest of this study Only three other studies found similarly largeproportions of aspartic acid (Ivarson and Sowden 1969 Read and

Bajwa 1985 Kielland 1995) Several amino acids were found in lowproportions across all studies isoleucine methionine phenylala-nine tryptophan and tyrosine The similar amino acid compositionacross a variety of different soils is consistent with reviews byAbuarghub and Read (1988) and Schulten and Schnitzer (1998)

There are only a handful of studies describing the amino acidcomposition in soil but the majority of laboratory and field studiesof amino acid uptake have focused on glycine Results of thisstudy indicate that glycine accounted for only 3 of the TFAA pool(Table 4) Thus we suggest that the influence of specific aminoacids to plant nutrition would be strengthened by further exami-nation of the abundance of these individual amino acids in the soil

42 Soil amino acid concentration cross succession

Amino acid concentrations generally increased across thesuccessional sequence (Fig 1b and c) probably due simply to theaccumulation of soil organic matter (Tables 1 and 2 Viereck et al1993a) which is a source of amino acids In addition boreal forestsoils high in organic matter also have high fungal biomass

1216 NR Werdin-Pfisterer et at Soil Biology amp Biochemistry 41 (2009) 1210-1220

(F1anafan and Van Cleve 1983) which is another source of aminoacids Kielland et al (2007) found that soil protease activityincreased across succession on the same plots sampled in this studyand suggested that the high concentrations of soil amino acidsin the later successional stages were sustained through highproteolytic activity

As organic matter accumulates across succession there is anassociated increase in cation exchange capacity (Van Cleve et al1993a) so more amino acids may be adsorbed onto the soilexchange sites of the later successional stages The basic aminoacid histidine should develop a strong positive charge in the lowpH conditions of late successional soils so the increase in histi-dine proportion and concentration over succession (Fig 1a-c)may reflect strong adsorption onto a greater number of soilexchange sites in the later successional stages Howeverproportions and concentrations of the other basic amino acidsarginine and lysine did not consistently increase across succes-sion (Table 3) so an explanation for the increased histidine

proportion and concentration remains uncertain A similarobservation was reported for heathland soils (Abuarghub andRead 1988)

Another possible explanation for the increased concentration ofsoil amino acids across succession is that the later successionalstages have greater belowground allocation in the form of fine roots(Ruess et al 2006) In the boreal forest belowground inputs fromroot turnover are greater and decompose faster than the above-ground litter inputs (Ruess et al 1996) The large spike in propor-tion and concentration of glutamine in the black sprucesuccessional stage (Fig 1 a and b) may result from greater quantitiesof root exudation or lysis from this active root biomass The patternof glutamine proportion and glutamine concentration per unit areaacross succession (Fig1 a and c) mirrors the pattern of below-ground fine root production across succession that Ruess et al(2006) measured using minirhizotrons We found high proportionsand concentrations of glutamine in the willow and black sprucestages where Ruess et al (2006) found the greatest allocation of

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1217

belowground production whereas we found a low proportion andconcentration of glutamine in the balsam poplar stage that had thelowest belowground allocation

43 Seasonal dynamics of soil amino acids

The composition and concentration of the soil amino acid poolappeared relatively constant over the growing season for eachsuccessional stage in contrast to our original hypothesis anddespite major seasonal changes in plant productivity litterfallmicrobial freeze-thaw flushes and soil temperatures Most studieshave found that amino acid concentrations do vary significantlyacross the season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005 though seeNordin et al 2001 Jones et al 2005a) Detecting seasonal changesif present may be problematic given that soil amino acids canfluctuate from high to low concentrations in less than a week(Weintraub and Schimel 2005) We sampled soils over three

months at monthly intervals which may constitute a rather coarsetemporal resolution

44 TFAA concentration compared to other studies

The range of TFAA concentrations of this study is within therange of values reported for a variety of ecosystems and climatesTable 4) Our mean TFAA values (20-350 nmol amino acid g 1 drysoil 04-54 ug amino acid N g 1 dry soil or 40-300 um) are similaror slightly higher than soil TFAA concentrations in the boreal forestin Sweden (5-455 nmol amino acid g 1 dry soil Persson and Nasholm 2001) the alpine dry meadows of Colorado (03-30 ug amino acid Ng-l dry soil Lipson et al 1999) and the mixedconifer and pygmy forests in northern California (16-299 um Yuet al 2002) We caution however about making inferences fromthese comparisons due to differences in soil sampling depthorganic matter content and extraction methods among the studies(Jones and Willett 2006)

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

1214 NR Werden-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220

acid histidine and TFAA concentrations on an areal basis across thesuccessional sequence TFAA concentration increased acrosssuccession ranging from 70 mg N m-2 for willow to 104 mg N m-2

for black spruce and TFAA concentration was 15 times greater inblack spruce than in willow Glutamine concentrations weresignificantly greater in black spruce than in alder balsam poplarand white spruce Glutamic acid concentrations were significantlylower in the shrub successional stages than in the tree stages(Plt 0001) Histidine concentrations increased across successionwhen calculated on a soil areal basis similar to the pattern ofincreasing histidine concentration across succession that wascalculated on a soil mass basis (Fig 1b)

32 Seasonal dynamics

321 Amino acid compositionComposition of the soil amino acid pool was fairly constant

across the growing season for each successional stage (Fig 2) Theonly trend was that proportions of glutamine were highest inSeptember for all successional stages Pairwise comparisonsshowed that glutamine proportions were significantly higher(Plt 005) in September than in August for the alder balsam poplarand white spruce stages

322 Amino acid concentrationConcentrations of soil amino acids in each successional stage

showed few clear and consistent patterns during the growingseason (Fig 3) Glutamic acid concentrations tended to be highestin June for the four earliest successional stages but the differenceswere not statistically significant The balsam poplar stage exhibited

the greatest variation in seasonal patterns of amino acid concen-trations In this successional stage twelve of the eighteen aminoacids (including the six dominant) had the highest concentrationsin June although these concentrations were not always statisticallysignificant Despite predictable variations in soil temperature andmoisture concentrations of TFAA showed no clear seasonalpatterns in any successional stage (Fig 4)

4 Discussion

41 Soil amino acid composition across succession

Despite major differences in terrace age vegetation inputsproductivity and soil properties the composition of the amino acidpool was remarkably similar in each of the five successional stagesAmino acid production and consumption in these soils entaila variety of sources (eg overstory and understory plant litter rootexudates animals and microbial cells) and processes (eg plantuptake microbial uptake and abiotic sorption) In addition theproduction and utilization of amino acids within plant or microbialcells as well as the ecosystem processes linking them likely sharethe same biochemistry and amino acid constituents regardless ofvegetation type soil environment or climate Although there arethese commonalities the capacity of plants and microbes to utilizeamino acids has been shown to vary among different plant speciesand functional types (Chapin et al 1993 Kielland 1994 Weigeltet al 2005) with amino acid molecular size (Kielland1994) aswell as being correlated with the concentration of the amino acidpool (Kielland 1994 Jones et al 2005a)

Elucidating the potential origin of amino acids in the soil iscomplicated due to the intertwined nature and speed of rhizo-sphere dynamics Analysis of amino acid composition of overstoryplant litter in these successional stages shows high proportions ofalanine glutamine and histidine similar to soil (unpublishedobservations) Other amino acids commonly found in arctic plantsinclude alanine arginine asparagine glutamine and glutamic acid(Chapin et aI 1986) the amino acid pool in boreal forest vegetation(shrub herb and grass foliage) is dominated by asparagine gluta-mine and arginine (Ohlson et al 1995) In addition to the aminoacids found in plant litter asparagine and glutamine are commonconstituents of plant xylem and phloem (Pate 1973 Kielland 1994Rochat 2001) whereas asparagine glutamine and arginine arecommon storage amino acids in above and belowground planttissue (Chapin et al 1986 Nasholm et al 1994 Ohlson et al 1995Nordin and Nasholm 1997) Microorganisms whose cell walls havehigh proportions of alanine aspartic acid and glutamic acid (Frie-del and Scheller 2002 Yu et al 2002) are also a likely source ofamino acids in the soil

A review of other studies demonstrates that there are commonsoil amino acids across a variety of natural and managed ecosys-tems from different climates further supporting the view that soilamino acids originate from similar ecosystem components orthrough similar biochemical processes (Table 4) Alanine and

NR Werdin-Pfisterer et a1 Soil Biology amp Biochemistry 41 (2009) 1210-1220 1215

glutamic acid are two common important amino acids acrossa variety of ecosystems Notably missing from our results were thelarge proportions of the amino acids arginine glycine and serinefound in nearly every other study including those in the borealforest in Sweden (Nordin et al 2001) and Alaskan tundra (Kielland1995) We did detect a large proportion (7) of arginine in the aldersuccessional stage perhaps because alder transports amino acidsfrom their N-fixing root nodules in the form of citrulline (Pate1973 Gaudillere 2001) a precursor to arginine In addition argi-nine is an efficient N storage compound (Chapin et al 1986) Nordinand Nasholm 1997) facilitating N storage during rapid N-fixation(Uliassi and Ruess 2002) Only a few studies report data forasparagine glutamine and histidine but these amino acids havebeen found to be important components of the amino acid pool(Ivarson and Sowden 1969 Abuarghub and Read 1988) Nordinet al 2001 Yu et al 2002) The amides asparagine and glutaminecombined made up 27 of the soil amino acid pool in both theboreal forest in Sweden (Nordin et al 2001) and the Alaskan borealforest of this study Only three other studies found similarly largeproportions of aspartic acid (Ivarson and Sowden 1969 Read and

Bajwa 1985 Kielland 1995) Several amino acids were found in lowproportions across all studies isoleucine methionine phenylala-nine tryptophan and tyrosine The similar amino acid compositionacross a variety of different soils is consistent with reviews byAbuarghub and Read (1988) and Schulten and Schnitzer (1998)

There are only a handful of studies describing the amino acidcomposition in soil but the majority of laboratory and field studiesof amino acid uptake have focused on glycine Results of thisstudy indicate that glycine accounted for only 3 of the TFAA pool(Table 4) Thus we suggest that the influence of specific aminoacids to plant nutrition would be strengthened by further exami-nation of the abundance of these individual amino acids in the soil

42 Soil amino acid concentration cross succession

Amino acid concentrations generally increased across thesuccessional sequence (Fig 1b and c) probably due simply to theaccumulation of soil organic matter (Tables 1 and 2 Viereck et al1993a) which is a source of amino acids In addition boreal forestsoils high in organic matter also have high fungal biomass

1216 NR Werdin-Pfisterer et at Soil Biology amp Biochemistry 41 (2009) 1210-1220

(F1anafan and Van Cleve 1983) which is another source of aminoacids Kielland et al (2007) found that soil protease activityincreased across succession on the same plots sampled in this studyand suggested that the high concentrations of soil amino acidsin the later successional stages were sustained through highproteolytic activity

As organic matter accumulates across succession there is anassociated increase in cation exchange capacity (Van Cleve et al1993a) so more amino acids may be adsorbed onto the soilexchange sites of the later successional stages The basic aminoacid histidine should develop a strong positive charge in the lowpH conditions of late successional soils so the increase in histi-dine proportion and concentration over succession (Fig 1a-c)may reflect strong adsorption onto a greater number of soilexchange sites in the later successional stages Howeverproportions and concentrations of the other basic amino acidsarginine and lysine did not consistently increase across succes-sion (Table 3) so an explanation for the increased histidine

proportion and concentration remains uncertain A similarobservation was reported for heathland soils (Abuarghub andRead 1988)

Another possible explanation for the increased concentration ofsoil amino acids across succession is that the later successionalstages have greater belowground allocation in the form of fine roots(Ruess et al 2006) In the boreal forest belowground inputs fromroot turnover are greater and decompose faster than the above-ground litter inputs (Ruess et al 1996) The large spike in propor-tion and concentration of glutamine in the black sprucesuccessional stage (Fig 1 a and b) may result from greater quantitiesof root exudation or lysis from this active root biomass The patternof glutamine proportion and glutamine concentration per unit areaacross succession (Fig1 a and c) mirrors the pattern of below-ground fine root production across succession that Ruess et al(2006) measured using minirhizotrons We found high proportionsand concentrations of glutamine in the willow and black sprucestages where Ruess et al (2006) found the greatest allocation of

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1217

belowground production whereas we found a low proportion andconcentration of glutamine in the balsam poplar stage that had thelowest belowground allocation

43 Seasonal dynamics of soil amino acids

The composition and concentration of the soil amino acid poolappeared relatively constant over the growing season for eachsuccessional stage in contrast to our original hypothesis anddespite major seasonal changes in plant productivity litterfallmicrobial freeze-thaw flushes and soil temperatures Most studieshave found that amino acid concentrations do vary significantlyacross the season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005 though seeNordin et al 2001 Jones et al 2005a) Detecting seasonal changesif present may be problematic given that soil amino acids canfluctuate from high to low concentrations in less than a week(Weintraub and Schimel 2005) We sampled soils over three

months at monthly intervals which may constitute a rather coarsetemporal resolution

44 TFAA concentration compared to other studies

The range of TFAA concentrations of this study is within therange of values reported for a variety of ecosystems and climatesTable 4) Our mean TFAA values (20-350 nmol amino acid g 1 drysoil 04-54 ug amino acid N g 1 dry soil or 40-300 um) are similaror slightly higher than soil TFAA concentrations in the boreal forestin Sweden (5-455 nmol amino acid g 1 dry soil Persson and Nasholm 2001) the alpine dry meadows of Colorado (03-30 ug amino acid Ng-l dry soil Lipson et al 1999) and the mixedconifer and pygmy forests in northern California (16-299 um Yuet al 2002) We caution however about making inferences fromthese comparisons due to differences in soil sampling depthorganic matter content and extraction methods among the studies(Jones and Willett 2006)

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

NR Werdin-Pfisterer et a1 Soil Biology amp Biochemistry 41 (2009) 1210-1220 1215

glutamic acid are two common important amino acids acrossa variety of ecosystems Notably missing from our results were thelarge proportions of the amino acids arginine glycine and serinefound in nearly every other study including those in the borealforest in Sweden (Nordin et al 2001) and Alaskan tundra (Kielland1995) We did detect a large proportion (7) of arginine in the aldersuccessional stage perhaps because alder transports amino acidsfrom their N-fixing root nodules in the form of citrulline (Pate1973 Gaudillere 2001) a precursor to arginine In addition argi-nine is an efficient N storage compound (Chapin et al 1986) Nordinand Nasholm 1997) facilitating N storage during rapid N-fixation(Uliassi and Ruess 2002) Only a few studies report data forasparagine glutamine and histidine but these amino acids havebeen found to be important components of the amino acid pool(Ivarson and Sowden 1969 Abuarghub and Read 1988) Nordinet al 2001 Yu et al 2002) The amides asparagine and glutaminecombined made up 27 of the soil amino acid pool in both theboreal forest in Sweden (Nordin et al 2001) and the Alaskan borealforest of this study Only three other studies found similarly largeproportions of aspartic acid (Ivarson and Sowden 1969 Read and

Bajwa 1985 Kielland 1995) Several amino acids were found in lowproportions across all studies isoleucine methionine phenylala-nine tryptophan and tyrosine The similar amino acid compositionacross a variety of different soils is consistent with reviews byAbuarghub and Read (1988) and Schulten and Schnitzer (1998)

There are only a handful of studies describing the amino acidcomposition in soil but the majority of laboratory and field studiesof amino acid uptake have focused on glycine Results of thisstudy indicate that glycine accounted for only 3 of the TFAA pool(Table 4) Thus we suggest that the influence of specific aminoacids to plant nutrition would be strengthened by further exami-nation of the abundance of these individual amino acids in the soil

42 Soil amino acid concentration cross succession

Amino acid concentrations generally increased across thesuccessional sequence (Fig 1b and c) probably due simply to theaccumulation of soil organic matter (Tables 1 and 2 Viereck et al1993a) which is a source of amino acids In addition boreal forestsoils high in organic matter also have high fungal biomass

1216 NR Werdin-Pfisterer et at Soil Biology amp Biochemistry 41 (2009) 1210-1220

(F1anafan and Van Cleve 1983) which is another source of aminoacids Kielland et al (2007) found that soil protease activityincreased across succession on the same plots sampled in this studyand suggested that the high concentrations of soil amino acidsin the later successional stages were sustained through highproteolytic activity

As organic matter accumulates across succession there is anassociated increase in cation exchange capacity (Van Cleve et al1993a) so more amino acids may be adsorbed onto the soilexchange sites of the later successional stages The basic aminoacid histidine should develop a strong positive charge in the lowpH conditions of late successional soils so the increase in histi-dine proportion and concentration over succession (Fig 1a-c)may reflect strong adsorption onto a greater number of soilexchange sites in the later successional stages Howeverproportions and concentrations of the other basic amino acidsarginine and lysine did not consistently increase across succes-sion (Table 3) so an explanation for the increased histidine

proportion and concentration remains uncertain A similarobservation was reported for heathland soils (Abuarghub andRead 1988)

Another possible explanation for the increased concentration ofsoil amino acids across succession is that the later successionalstages have greater belowground allocation in the form of fine roots(Ruess et al 2006) In the boreal forest belowground inputs fromroot turnover are greater and decompose faster than the above-ground litter inputs (Ruess et al 1996) The large spike in propor-tion and concentration of glutamine in the black sprucesuccessional stage (Fig 1 a and b) may result from greater quantitiesof root exudation or lysis from this active root biomass The patternof glutamine proportion and glutamine concentration per unit areaacross succession (Fig1 a and c) mirrors the pattern of below-ground fine root production across succession that Ruess et al(2006) measured using minirhizotrons We found high proportionsand concentrations of glutamine in the willow and black sprucestages where Ruess et al (2006) found the greatest allocation of

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1217

belowground production whereas we found a low proportion andconcentration of glutamine in the balsam poplar stage that had thelowest belowground allocation

43 Seasonal dynamics of soil amino acids

The composition and concentration of the soil amino acid poolappeared relatively constant over the growing season for eachsuccessional stage in contrast to our original hypothesis anddespite major seasonal changes in plant productivity litterfallmicrobial freeze-thaw flushes and soil temperatures Most studieshave found that amino acid concentrations do vary significantlyacross the season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005 though seeNordin et al 2001 Jones et al 2005a) Detecting seasonal changesif present may be problematic given that soil amino acids canfluctuate from high to low concentrations in less than a week(Weintraub and Schimel 2005) We sampled soils over three

months at monthly intervals which may constitute a rather coarsetemporal resolution

44 TFAA concentration compared to other studies

The range of TFAA concentrations of this study is within therange of values reported for a variety of ecosystems and climatesTable 4) Our mean TFAA values (20-350 nmol amino acid g 1 drysoil 04-54 ug amino acid N g 1 dry soil or 40-300 um) are similaror slightly higher than soil TFAA concentrations in the boreal forestin Sweden (5-455 nmol amino acid g 1 dry soil Persson and Nasholm 2001) the alpine dry meadows of Colorado (03-30 ug amino acid Ng-l dry soil Lipson et al 1999) and the mixedconifer and pygmy forests in northern California (16-299 um Yuet al 2002) We caution however about making inferences fromthese comparisons due to differences in soil sampling depthorganic matter content and extraction methods among the studies(Jones and Willett 2006)

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

1216 NR Werdin-Pfisterer et at Soil Biology amp Biochemistry 41 (2009) 1210-1220

(F1anafan and Van Cleve 1983) which is another source of aminoacids Kielland et al (2007) found that soil protease activityincreased across succession on the same plots sampled in this studyand suggested that the high concentrations of soil amino acidsin the later successional stages were sustained through highproteolytic activity

As organic matter accumulates across succession there is anassociated increase in cation exchange capacity (Van Cleve et al1993a) so more amino acids may be adsorbed onto the soilexchange sites of the later successional stages The basic aminoacid histidine should develop a strong positive charge in the lowpH conditions of late successional soils so the increase in histi-dine proportion and concentration over succession (Fig 1a-c)may reflect strong adsorption onto a greater number of soilexchange sites in the later successional stages Howeverproportions and concentrations of the other basic amino acidsarginine and lysine did not consistently increase across succes-sion (Table 3) so an explanation for the increased histidine

proportion and concentration remains uncertain A similarobservation was reported for heathland soils (Abuarghub andRead 1988)

Another possible explanation for the increased concentration ofsoil amino acids across succession is that the later successionalstages have greater belowground allocation in the form of fine roots(Ruess et al 2006) In the boreal forest belowground inputs fromroot turnover are greater and decompose faster than the above-ground litter inputs (Ruess et al 1996) The large spike in propor-tion and concentration of glutamine in the black sprucesuccessional stage (Fig 1 a and b) may result from greater quantitiesof root exudation or lysis from this active root biomass The patternof glutamine proportion and glutamine concentration per unit areaacross succession (Fig1 a and c) mirrors the pattern of below-ground fine root production across succession that Ruess et al(2006) measured using minirhizotrons We found high proportionsand concentrations of glutamine in the willow and black sprucestages where Ruess et al (2006) found the greatest allocation of

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1217

belowground production whereas we found a low proportion andconcentration of glutamine in the balsam poplar stage that had thelowest belowground allocation

43 Seasonal dynamics of soil amino acids

The composition and concentration of the soil amino acid poolappeared relatively constant over the growing season for eachsuccessional stage in contrast to our original hypothesis anddespite major seasonal changes in plant productivity litterfallmicrobial freeze-thaw flushes and soil temperatures Most studieshave found that amino acid concentrations do vary significantlyacross the season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005 though seeNordin et al 2001 Jones et al 2005a) Detecting seasonal changesif present may be problematic given that soil amino acids canfluctuate from high to low concentrations in less than a week(Weintraub and Schimel 2005) We sampled soils over three

months at monthly intervals which may constitute a rather coarsetemporal resolution

44 TFAA concentration compared to other studies

The range of TFAA concentrations of this study is within therange of values reported for a variety of ecosystems and climatesTable 4) Our mean TFAA values (20-350 nmol amino acid g 1 drysoil 04-54 ug amino acid N g 1 dry soil or 40-300 um) are similaror slightly higher than soil TFAA concentrations in the boreal forestin Sweden (5-455 nmol amino acid g 1 dry soil Persson and Nasholm 2001) the alpine dry meadows of Colorado (03-30 ug amino acid Ng-l dry soil Lipson et al 1999) and the mixedconifer and pygmy forests in northern California (16-299 um Yuet al 2002) We caution however about making inferences fromthese comparisons due to differences in soil sampling depthorganic matter content and extraction methods among the studies(Jones and Willett 2006)

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210-1220 1217

belowground production whereas we found a low proportion andconcentration of glutamine in the balsam poplar stage that had thelowest belowground allocation

43 Seasonal dynamics of soil amino acids

The composition and concentration of the soil amino acid poolappeared relatively constant over the growing season for eachsuccessional stage in contrast to our original hypothesis anddespite major seasonal changes in plant productivity litterfallmicrobial freeze-thaw flushes and soil temperatures Most studieshave found that amino acid concentrations do vary significantlyacross the season (Abuarghub and Read 1988 Kielland 1995Lipson et al 1999 Weintraub and Schimel 2005 though seeNordin et al 2001 Jones et al 2005a) Detecting seasonal changesif present may be problematic given that soil amino acids canfluctuate from high to low concentrations in less than a week(Weintraub and Schimel 2005) We sampled soils over three

months at monthly intervals which may constitute a rather coarsetemporal resolution

44 TFAA concentration compared to other studies

The range of TFAA concentrations of this study is within therange of values reported for a variety of ecosystems and climatesTable 4) Our mean TFAA values (20-350 nmol amino acid g 1 drysoil 04-54 ug amino acid N g 1 dry soil or 40-300 um) are similaror slightly higher than soil TFAA concentrations in the boreal forestin Sweden (5-455 nmol amino acid g 1 dry soil Persson and Nasholm 2001) the alpine dry meadows of Colorado (03-30 ug amino acid Ng-l dry soil Lipson et al 1999) and the mixedconifer and pygmy forests in northern California (16-299 um Yuet al 2002) We caution however about making inferences fromthese comparisons due to differences in soil sampling depthorganic matter content and extraction methods among the studies(Jones and Willett 2006)

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

1218 NR Werdin-Pfisterer et al Soil Biology Biochemistry 41 (2009) 1210-1220

5 Conclusions

Contrary to our original hypothesis the composition of the soilamino acid pool did not change across succession Irrespective ofsuccessional stage the soil amino acid pool was dominated by thesame six amino acids glutamic acid glutamine aspartic acidasparagine alanine and histidine However the concentration ofsoil amino acids did significantly increase across the successionalsequence and amino acid concentrations were an order of

magnitude higher in the coniferous-dominated late successionalstages than in the early deciduous-dominated stages In additiondespite major seasonal changes in plant productivity Iitterfallmicrobial freeze-thaw flushes and soil temperature the compo-sition and concentration of the soil amino acid pool were generallyconstant over the growing season for each successional stage Thesimilar amino acid composition across the successional sequencesuggests that amino acids originate from a common source orthrough similar biochemical processes yet elucidating which

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

NR Werdin-Pfisterer et al Soil Biology amp Biochemistry 41 (2009) 1210~1220 1219

ecosystem components or processes regulate a particular aminoacid needs further investigation Results of this study demonstratethat amino acids are important constituents of the biogeochemi-cally diverse soil N pool in the boreal forest of interior Alaska

Acknowledgements

Special thanks to Melinda Brunner and Karl Olson for field andlaboratory assistance Susan Henrichs and Donald Schell for use oftheir HPLC and Rolf Gradinger for HPLC laboratory space We alsoare indebted to Roger Ruess and two anonymous reviewers forhelpful comments on earlier versions of this manuscript Thisresearch was funded by a United States Department of AgricultureEcosystems grant with additional support from the Bonanza CreekLong Term Ecological Research program (funded jointly by NSF andUSDA Forest Service Pacific Northwest Research Station)

References

Abuarghub SM Read OJ 1988 The biology of mycorrhiza in the Ericaceae XIIQuantitative analysis of individual free amino acids in relation to time anddepth in the soil profile New Phytologist 108 433-441

Anderson GS 1970 Hydrologic Reconnaissance of the Tanana Basin CentralAlaska Us Geological Survey Hydrological Investigations HA319

Bonanza Creek LTER Database 2002a Bonanza Creek Experimental Forest ActiveLayer Depths at Core Floodplain Sites Bonanza Creek Long Term EcologicalResearch Program Fairbanks AK httpwwwlteruafedudata detailcfmdatafilepkey=ll

Bonanza Creek LTER Database 2002b Bonanza Creek Experimental Forest HourlySoil Temperature at Varying Depths from 1988 to Present Bonanza Creek LongTerm Ecological Research Program Fairbanks AK httpwwwlteruafedudatadetailcfmdatafile pkey 3

Chapin III FS Kedrowski RA 1983 Seasonal changes in nitrogen and phosphorusfractions and autumn retranslocation in evergreen and deciduous taiga treesEcology 64 376-391

Chapin III FS Shaver GR Kedrowski RA 1986 Environmental controls overcarbon nitrogen and phosphorus fractions in Eriophorum voginowm in Alaskantussock tundra journal of Ecology 74 167-195

Chapin III FS Moilanen L KielJand 1lt1993 Preferential use of organic nitrogenfor growth by a non-mycorrhizal arctic sedge Nature 631 150-153

Collins CM 1990 Morphometric Analyses of Recent Channel Changes on theTanana River in the Vicinity of Fairbanks Alaska llS Army Corps of EngineersCold Regions Research and Engineering laboratory Hanover NH CRRELReport90-448 pp

Flanagan PW Van Cleve K 1983 Nutrient cycling in relation to decompositionand organic-matter quality in taiga ecosystems Canadian journal of ForestResearch 13 795-817

FriedeljK Scheller E 2002 Composition ofhydrolysable amino acids in soil organicmatter and soil microbial biomass Soil Biology amp Biochemistry 34 315-325

Caudillere [P 2001 Management of nitrogen in ligneous species In Morot-Gaudry j-F (Ed) Nitrogen Assimilation by Plants Physiological Biochemicaland Molecular Aspects Science Publishers lnc Enfield NH pp 331-341

Hatcher L Stepanski EJ 1994 A Step-by-Step Approach to using the SAS Systemfor Univariate and Multivariate Statistics SAS Institute Inc Cary NC 552 pp

Hendershot WH Lalande H Duquette M 1993 Soil reaction and exchangeacidity In Carter MR (Ed) Soil Sampling and Methods of Analysis CanadianSociety of Soil Science Lewis Publishers Boca Raton FL pp 141-145

Ivarson ICC Sowden Fj 1966 Effect of freezing on the free amino acids in soilCanadian journal of Soil Science 46 115- 120

Ivarson KC Sowden FJ1969 Free amino acid composition of the plant root envi-ronment under field conditions Canadian journal of Soil Science 49 121-127

jarret HW Cooksy KD Ellis B Anderson jM 1986 The separation of 0-

phthalaldehyde derivatives of amino acids by reversed-phase chromatographyon octylsilica columns Analytical Biochemistry 153 189-198

jones DL 1999 Amino acid biodegradation and its potential effects on organicnitrogen capture by plants Soil Biology amp Biochemistry 31 613-622

jones DL Willett VB 2006 Experimental evaluation of methods to quantifydissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soilSoil Biology amp Biochemistry 38 991-999

jones DL Shannon D junvee-Fortune T Farrar jF 2005a Plant capture of freeamino acids is maximized under high soil amino acid concentrations SoilBiology amp Biochemistry 37 179-181

jones DL Healey jR Willett VB Farrar jF Hodge A 2005b Dissolved organicnitrogen uptake by plants - an important N uptake pathway Soil Biology ampBiochemistry 37 413-423

Kielland K 1994 Amino acid absorption by arctic plants implications for plantnutrition and nitrogen cycling Ecology 75 2373-2383

Kielland 1lt 1995 Landscape patterns of free amino acids in arctic tundra soilsBiogeochemistry 32 1-14

Kielland K 2001 Short-circuiting the nitrogen cycle strategies of nitrogen uptakein plants from marginal ecosystems In Ae N Arihara j Okada KSrinivasan A (Eds) Plant Nutrient Acquisition New Perspectives Springer-Verlag Berlin pp 376-398

KieJland K McFarland jW Ruess RW Olson Kbull 2007 Rapid cycling of organicnitrogen in taiga forest ecosystems Ecosystems 10 360- 368

Lipson DA Monson RK 1998 Plant-microbe competition for soil amino acids in thealpine tundra effects of freeze-thaw and dry-rewetevents Oecolngia 113406-414

Lipson 0Nasholrn T 2001 The unexpected versatility of plants organic nitrogenuse and availability in terrestrial ecosystems Oecologia 128305-316

Lipson DA Schmidt SK Monson RIlt1999 links between microbial populationdynamics and nitrogen availability in an alpine ecosystem Ecology SO 1623-1631

Littell RC Stroup Ww Freund RJ 2002 SAS for Linear Models fourth ed SASInstitute Inc Cary NC 466 pp

Nasholrn T Edfast A-B Ericsson A Norden L-G 1994 Accumulation of aminoacids in some boreal forest plants in response to increased nitrogen availabilityNew Phytologisr 126 137-143

Nasholrn T Ekblad A Nordin A Giesler R Hogberg M Hogberg 1 1998 BorealForest plants take up organic nitrogen Nature 392914-916

NADP National Atmospheric Deposition ProgramNational Trends Network 20022001 Annual and Seasonal Data Summary for Site AKOI NADPNTN ProgramOffice Champaign IL httpnadpsw5uiuceduads2001ak01pdl

Nordin A Nasholm T 1997 Nitrogen storage forms in nine boreal undersroreyplant species Oecologia 110487-492

Nordin A Hogberg P Nasholrn T 2001 Soil nitrogen form and plant nitrogenuptake along a boreal torest productivity gradient Oecologia 129 125- 132

Ohlson M Nordin A Nasholrn T 1995 Accumulation of amino acids in forestplants in relation to ecological amplitude and nitrogen supply FunctionalEcology 9 596-605

Pate jS 1973 Uptake assimilation and transport of nitrogen compounds by plantsSoil Biology amp Biochemistry 5 109-119

Persson j Nasholm T 2001 Amino acid uptake a widespread ability amongboreal forest plants Ecology Letters 4 434-438

Ping CL johnson AX 2000 Soil Horizon DescriptionsClassification and LabAnalysis Bonanza Creek Long Term Ecological Research Program Fairbanks AKhttplteruafedudatafile pkey 149

Raab TK Lipson DA Monson RIC 1996 Non-mycorrhizal uptake of amino acidsby roots of the alpine sedge Kobiesia myosuroides implications for the alpinenitrogen cycle Oecologia 108 488-494

Read Dj Bajwa R 1985 Some nutritional aspects of the biology of ericaceousmycorrhizas Proceedings of the Royal Society of Edinburgh 85B 317-332

Read Dj Perez-Moreno j 2003 Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance New Phytologist 157 475-492

Rochat C 2001 Transport of amino acids in plants In Morot-Caudry J-F (Ed)Nitrogen Assimilation by Plants Physiological Biochemical and MolecularAspects Science Publishers Inc Enfield NH pp 253-267

Roth M 1971 Fluorescence reaction tor amino acids Analytical Chemistry 43 880-882Ruess RW Van Cleve K Yarie j Viereck LA 1996 Contributions of fine root

production and turnover to the carbon and nitrogen cycling in taiga forests ofthe Alaskan interior Canadian journal of Forest Research 26 1326- 1336

Ruess Rw Hendrick RL Vogel JC Sveinbjornsson B 2006 The role of fineroots in the functioning of boreal forests In Chapin III FS Oswood MW VanCleve K Viereck LA Verbyla DL (Eds) Alaskas Changing Boreal ForestOxford University Press New York NY pp 189-210

Schimel [P Chapin III FS 1996 Tundra plant uptake of arnino acid and NH4nitrogen in situ plants compete well for amino acid N Ecology 77 2142-2147

Schulten HR Schnitzer M 1998 The chemistry of soil organic nitrogen a reviewBiology and Ferti lity of Soils 26 1- 15

SYSTAT Software lnc 2002 PeakFit 411 for Windows Users Manual SYSTATSoftware Inc Richmond CA

Uliassi DO Ruess RW 2002 limitations to symbiotic nitrogen fixation inprimary succession on the Tanana River floodplain Ecology 83 88-103

Van Cleve K Alexander V 1981 Nitrogen cycling in tundra and boreal ecosystemsIn Clark FE Rosswall T (Eds) Terrestrial Nitrogen Cyctes Ecological Bulletins33 375-404

Van Cleve K Chapin III Fs Dyrness CT Viereck LA 1991 Element cycling intaiga forests state factor control Bioscience 41 78-88

Van Cleve K Dyrness CT Marion GM Erickson R 1993a Control of soildevelopment on the Tanana River floodplain interior Alaska Canadian Journalof Forest Research 23 941-955

Van Cleve K Viereck lA Marion CM 1993b Introduction and overview ofa study dealing with the role of salt-affected soils in primary succession on theTanana River floodplain interior Alaska Canadian journal afForest Research 23879-888

Van Cleve K YarieJ Erickson R Dyrness CT 1993c Nitrogen mineralization andnitrification in successional ecosystems on tile Tanana River floodplain interiorAlaska Canadian journal of Forest Research 23 970-978

Viereck lA 1989 Flood-plain succession and vegetation classification in interiorAlaska In Ferguson DE Morgan P johnson FD (Eds) Proceedings LandClassifications Based on Vegetation Applications lor Resource Management LlSDepartment of Agriculture Forest Service Pacific Northwest Forest and RangeExperimentStation Portland OR pp 197-202 Ceneral Technical Report INT-257

Viereck LA Dyrness CT Foote MJ 1993a An overview of tile vegetation ancisoils of tile floodplain ecosystems of the Tanana River interior Alaska Canadianjournal of Forest Research 23 889-898

1220 NR Werdin-~fistmr et al Soil Biology [7 BiociJel11istry 41 (2009) 1210-1220

Viereck LA Van Cleve K Adams Pc Schletuer RE 1993b Climate of the TananaRiver floodplain near Fairbanks Alalka Canadian Journal of Forest Research 23891-913

Weigel A 801 R Bardgett RD 2005 Preferential uptake of soil nitrogen forms bygrassland species Oecologia 142627-635

Weintraub MN Schimel jP 2005 The seasonal dynamics of amino acidsand other nutrients in Alaskan Arctic tundra soils Biogeochemistry 73359-380

Varie J Van Cleve K Dyrness CT Oliver L Levison I Erickson R 1993 Soil-solution chemistry in relation to forest succession on the Tanana River flood-plain interior Alaska Canadian Journal of Forest Research 23 928-940

Yarie] Viereck L Van Cleve K Adams P 1998 Flooding and ecosystem dynamicsalong the Tanana River Bioscience 48 690-695

Yu Z Zhang Q Kraus TEC Dahlgren KA Anastasio C Zasoski RJ 2002Contribution of amino compounds to dissolved organic nitrogen in forest soilsBiogeochemistry 61 173-198