Soil amino acid utilization among species of the Cyperaceae: Plant and Soil Processes

Transcription

1 Stanfor University From the SeleteWorks of Te K. Raab Summer 1999 Soil amino ai utilization among speies of the Cyperaeae: Plant an Soil Proesses Te K. Raab, Stanfor University Davi A. Lipson, Carnegie Institution of Washington Steven K. Smit, University of Colorao at Bouler Russ K. Monson, University of Arizona Available at:

2 Eology, 80(7), 1999, pp by the Eologial Soiety of Ameria SOIL AMINO ACID UTILIZATION AMONG SPECIES OF THE CYPERACEAE: PLANT AND SOIL PROCESSES THEODORE K. RAAB, DAVID A. LIPSON, AND RUSSELL K. MONSON 1 Department of Environmental, Population an Organismi Biology, University of Colorao, Bouler, Colorao USA Abstrat. Amino ais are release uring the eomposition of soil organi matter an have been shown to be utilize as a nitrogen soure by some non-myorrhizal speies in the family Cyperaeae (the sege family). Twelve out of 13 Cyperaeae speies examine in the urrent stuy were apable of absorbing soil amino ais in the non-myorrhizal state. With two exeptions (two speies in the genus Kobresia), speies from subalpine or alpine habitats exhibite lower rates of total nitrogen uptake ompare to speies from more temperate habitats, whih is possibly explaine by lower growth rates in the alpine an subalpine speies an a lower overall eman for soil nitrogen. The alpine an subalpine speies exhibite higher rates of glyine uptake relative to NH 4 an NO 3 uptake, ompare to speies from the more temperate habitats. This may reflet speialization towar the uptake of organi N in the alpine an subalpine speies. In three eosystems where seges ommonly our, amino ais were present in the soil pore water, but in highly variable amounts. Seasonal maximum amino ai onentrations in alpine soil pore water range from 13 to 158 mol/l in four ry meaow sites. The most ommon amino ai in these alpine soils was glyine. In a subalpine fen habitat, aspartate was the most ommonly observe amino ai, an total amino ai onentrations were mol/l. In a shortgrass steppe habitat, glutamate was the most abunant amino ai, an total amino ai onentrations were mol/l. Soil protease ativities orrelate positively with soil protein ontent when all three eosystems (alpine, subalpine fen, an shortgrass steppe) were onsiere together. The aition of protein (as asein) signifiantly inrease soil protease ativity, iniating that soil protein ontent ontributes to the apaity to generate soil amino ais. Soil protein ontents orrelate positively with total soil N ontent when the alpine an shortgrass steppe sites were onsiere together. The retention of NH 4 an amino ais on the alpine soil was high ompare to soils of lower organi matter ontent that have been investigate in past stuies. We onlue that (1) the potential to take up amino ais in non-myorrhizal roots of the family Cyperaeae is wiesprea among speies native to a variety of ifferent habitats, (2) amino ais are available for uptake (though most abunant in the alpine), (3) the potential to generate soil amino ais is proportional to soil protein onentration, an (4) soil amino ais an be retaine in the soil at high levels, leaing to the onlusion that the soil bining affinity of amino ais may onstrain uptake by plant roots an miroorganisms. Key wors: alpine; amino ais; ammonium uptake; Carex; Cyperaeae; Cyperus; Kobresia; nitrate uptake; nutrient uptake; proteolysis; shortgrass steppe; subalpine fen. INTRODUCTION Through reent stuies, it has beome evient that soil amino ais are a prinipal soure of nitrogen for ertain plants. Though this has been known for sometime in myorrhizal, heathlan speies (Rea 1993), it is also a feature of non-myorrhizal plants from arti an alpine eosystems (Chapin et al. 1993, Kiellan 1994, 1997, Raab et al. 1996). These systems are similar in that N mineralization rates are heavily on- Manusript reeive 14 May 1998; revise 4 September 1998; aepte 22 September Aress reprint requests an orresponene to this author at Department of EPO Biology, Campus Box 334, University of Colorao, Bouler, Colorao USA straine by limate, an plant N emans annot be met through the uptake of inorgani ions. A number of issues remain to be eluiate onerning the utilization of soil amino ais by plants. First, it is not lear how wiesprea amino ai uptake is among speies native to habitats aross a wie range of soil organi matter ontent. Seon, it is not lear what ontrols amino ai onentrations in ertain soils. One an assume that aumulation iniates an imbalane between the proesses that supply amino ais (e.g., proteolysis by soil enzymes) an those that onsume or bin free amino ais (e.g., uptake by plants an miroorganisms an asorption to the soil). However, the relative magnitue of these proesses has not been assesse in past stuies.

3 Otober 1999 AMINO ACID AVAILABILITY TO SEDGES 2409 In the stuies esribe here, several aspets of soil amino ai utilization were explore. The apaity for non-myorrhizal amino ai uptake was stuie in 13 speies of the family Cyperaeae to isern whether this trait is wiesprea within a group of plants previously shown to exhibit the trait. In aition to measurements of amino ai uptake, measurements were mae for the uptake of NO 3 an NH 4, to etermine the maximal physiologial apaity for the uptake of organi N relative to inorgani N. For eight of the speies, whih are native to three ifferent eosystems in Colorao (alpine tunra, shortgrass steppe, an subalpine fen), the abunane an seasonal ynamis of soil amino ais were measure in soil pore water. This permitte us to test the hypothesis that soil amino ais are available for plant uptake in eosystems other than the arti an alpine. Finally, stuies were onute on the effet of soil protein onentration on proteolysis rate, interations of amino ais with the exhange omplex of the soil, an the ombine impliations of these proesses for amino ai availability. MATERIALS AND METHODS Stuy site esriptions The alpine stuies were onute in the Colorao Front Range at the Niwot Rige Biosphere Reserve (40 03 N, W; 3550 m elevation). The stuies were onute in ry meaow habitats, whih are blown free of snow uring muh of the winter, an are overe with ense tussoks of the irumpolar sege Kobresia myosuroies (Vill.) Fiori an Paol. The meaows are not subjet to the influene of permafrost. Soils at the site are lassifie as Pergeli Cryumbrepts an Pergeli Cryohrepts (Osburne an Cline 1959, Burns 1980, Birkelan et al. 1989). Mirolysimetry stuies were onute in four ifferent ry meaows at the Niwot Rige site. The meaows were separate by at least 0.2 km. Within eah meaow 10 mirolysimeters were installe at 1-m intervals along a linear transet. Climate ata for Niwot Rige have been publishe by Greenlan (1989). Stuies of a subalpine, minerotrophi fen were onute at High Creek Fen in Park County, Colorao (39 15 N, W; 3100 m elevation). Soils at the fen are lassifie as Histi Cryaquolls built on alluvium an glaial outwash (Leo Kott, Natural Resoures Conservation Servie, Cripple Creek, Colorao, personal ommuniation). Climate ata provie by the U.S. Forest Servie at Fairplay, Colorao (15 km N of the fen site) iniate a montane limate with mean annual preipitation of m, an mean annual temperature of 2 4 C. Two transets hosen for mirolysimeters our in an area of frequent hummoks (Hi Fen) ominate by Kobresia simpliiusula (Wahlenb.) Mak an Carex miroglohin Wahlenb., as well as a low-lying, wet meaow (Lo Fen) ominate by Eriophorum angustifolium Honk. an Carex aquatilis Wahlenb. Five mirolysimeters were installe at 1-m intervals along a linear transet at eah of these two fen sites. Stuies of the shortgrass steppe eosystem were onute at the Central Plains Experimental Range, in northeastern Colorao (40 42 N, W; 1640 m elevation). The stuies were onute at the base of a slope ontaine within an enlosure that has been protete from grazing sine The soils are lassifie as Pahi Argiustolls (Shimel et al. 1985). The site is ominate by Carex stenophylla Wahlenb. ssp. eleoharis (Bailey), Carex fillifolia Nutt., Agropyron smithii Ryb., Bouteloua grailis (H.B.K.) Lag, an Buhloe atyloies (Nutt.) Englem. Ten mirolysimeters were burie at 1-m intervals at this site. Three of the Cyperaeae speies that were stuie (Cyperus halpan L., Gahnia gahniformis (Gan.) A. Heller, an Carex wahuensis C.A. Mey) were ollete as roote in Hawai i Volanoes National Park (19 32 E, W, 1200 m elevation). This site is lassifie as tropial ry forest, with a more etaile esription provie in Ley an D Antonio (1998). Three aitional speies (Carex anesens L., Carex rhynhophysa C. A. Mey, an Cyperus strigosus L.) were grown from see obtaine from the USDA Western Regional Plant Introution Servie in Pullman, Washington. Mirolysimetry of the rooting zone an analysis of inorgani an organi N frations Mirolysimeters (Rhizosphere Researh Prouts, Wageningen, The Netherlans) were installe at 10-m epth in the A-horizon at all stuy sites. At least 4 mo elapse between the time of installation an solution sampling. Sampling was onute from May to Otober 1996 for the alpine an fen transets an from April to September 1996 for the shortgrass steppe transet. The mirolysimeters (10 m length, 2.3 mm iameter) are onstrute of a porous polymeri material that allows olletion of filtere (to 0.2 m) soil-pore water above tensions of 0.1 MPa. Using the Luerlok onnetions on the mirolysimeter for insertion of a hypoermi neele, soil water was ollete in 7 mlraw sterile, evauate olletion tubes (Terumo Venojet, Elkton, Marylan). As ollete, soil-pore water samples ha volumes ranging from 0.5 to 6 ml. Prior to freezing at 20 C for storage, the samples were ivie into ml aliquots (the exat volume epene on the intene assay protool) an plae into sterile Eppenorf mirotubes for the various analyses. Samples were measure within 72 h of olletion. Nitrate was assaye using EPA Metho (O Dell et al. 1984) on a Dionex 3500I ion hromatograph (Dionex Corporation, Sunnyvale, California) utilizing suppresse onutivity etetion, as esribe in Raab et al. (1996). Ammonium was assaye on a Moleular Devies (Moleular Devies Corporation, Sunnyvale, California) spe-

4 2410 THEODORE K. RAAB ET AL. Eology, Vol. 80, No. 7 TABLE 1. Ientifiation of seges use in N uptake stuies. Speies Soure Native ommunity 1. Kobresia myosuroies (Vill.) Fiori an Paol. 2. Carex rupestris All. 3. Carex albonigra Makenzie 4. Kobresia simpliiusula (Wahlenb.) Mak. 5. Carex ebenea Ryb. 6. Carex anesens L. 7. Carex rhynhophysa C.A. Mey 8. Carex stenophylla Wahlenb. ssp. eleoharis (Bailey) 9. Carex fillifolia Nutt. 10. Cyperus strigosus L. 11. Cyperus halpan L. 12. Gahnia gahniformis (Gan.) A. Heller 13. Carex wahuensis C.A. Mey see see see see alpine, Colorao alpine, Colorao alpine, Colorao subalpine fen, Colorao subalpine forest, Wyoming subalpine forest, Wyoming subalpine forest, Alaska shortgrass steppe, Colorao shortgrass steppe, Colorao temperate swamp, North Carolina ry tropial forest, Hawaii ry tropial forest, Hawaii ry tropial forest, Hawaii trophotometri plate reaer at 650 nm using the phenolhypohlorite hemistry of Solorzano (1969). Amino ais were measure using reverse-phase high pressure liqui hromatography (HPLC) on a Bekman System Gol (Bekman Coulter Inorporate, Fullerton, California) following erivatization with 4-imethylaminobenzene-4 -sulfonyl hlorie (DABS). Samples were separate on a thermostate (35 C) 25 m long C 18 olumn (5 m) using an aetate/methanol/ tetrahyrofuran graient (Chang et al. 1983). The relationships among speies aoring to ANOVA with respet to the uptake of ifferent nitrogen ompouns. TABLE 2. Speies Uptake Glyine Nitrate Ammonium b, e, e b,, e, e, e b, not appliable, e b,, a, not appliable, b,, a b Notes: See Table 1 for ientifiation of speies. Speies with the same letter(s) in a olumn are not statistially ifferent aoring to Tukey s Stuentize Range Test at P In two ases, the test results are labele as not appliable beause uptake of the ompoun was not observe. Nutrient-uptake measurements Plants use in the uptake experiments were establishe from see or utting, as iniate in Table 1. Sees were ol sarifie for 1 wk an sown in a soilless potting mix (2:2:1 of vermiulite/perlite/zeolite). Alternatively, of plants growing in the fiel were trimme to the root rown to eliminate existing leaves an roots. After an initial establishment in the greenhouse uner a misting apparatus, the resultant plants were grown in the growth hamber or greenhouse. Plants from the alpine eosystem were grown in an environmentally ontrolle growth hamber (Conviron, Ashville, North Carolina, moel E-15) programme to provie a photon flux ensity ( nm) of 550 mol m 2 s 1 an a 15 /9 C thermoperio with a 15-h photoperio, for 4 mo prior to measurement. Plants from the shortgrass steppe an subtropial eosystems, an the one speies from North Carolina, were grown in a glasshouse with temperature programme to C an natural photon flux ensities an photoperio. All plants were watere 1 2 times per week as neee with istille water, an fertilize one per week with one-eighth strength, moifie Hoaglan s solution as esribe in Raab et al. (1996). At the time of the nutrient uptake experiments, plants were remove from their potting mix, an the roots rinse with 50 mmol/l KCl to remove ahering partiles. Intat plants were suspene in a 1200 ml aerate, temperature-ontrolle hyroponi uvette ontaining a solution of 100 mol/l CaSO 4 (2H 2 O), an 125 mol/l of either KNO 3,NH 4 Cl, or glyine. All uptake solutions were prepare with 18 M (tissue ulture) grae water. Levels of [Ca 2 ] 100 mol/l are often reommene for hyroponi uptake experiments to insure root membrane integrity (Epstein 1961). The N onentration (125 mol/l) that was use for the uptake experiments was hosen as the highest level observe in fiel olletions from the mirolysimeters. Using a thermostate, irulating water bath that fore water through a jaket of opper tubing enirling the uvette (though not in ontat with the plant or solution), solution temperature was maintaine at 15 C for all speies. After a 2-h initial equilibration, samples of 1-mL volume were remove from the uvette at 1 2 h intervals over 24 h, an frozen until analyze. Amino ai onentration of the samples was measure using HPLC as esribe above. Ammonium was routinely etermine (but rarely observe) on amino ai uptake samples by the plate reaer metho esribe above. The root uvette was leane after eah experiment with 2.5% aqueous soium hypohlorite followe by rinses with eionize water. Aluminum

5 Otober 1999 AMINO ACID AVAILABILITY TO SEDGES 2411 foil an moeling lay limite light transmission to the root zone. Mirobial growth was never observe in the hyroponi system when using NH 4,NO 3, or glyine. However, preliminary experiments with glutamate emonstrate visible turbiity after 20 h, an thus was not onsiere further for uptake omparisons. Several other amino ais (e.g., glutamine an lysine) observe in the soil are also unstable (over h) in hyroponi experiments. (Our preliminary stuies iniate that glyine is a poor substrate for mirobial respiration or growth when inlue in native soil, or aqueous or gelatinous meia [D. A. Lipson, S. K. Shmit, R. K. Monson, unpublishe results].) In numerous previous experiments, measurements were onute to etermine if glyine in the root uvette was mineralize to NH 4 uring uptake measurements (Raab et al. 1996). In no ase has measurable mineralization been observe over the time ourse of the uptake experiments. Plant root growth was frequently observe through measure inreases in root length uring the ourse of several preliminary, long hyroponi experiments. Thus, there is no eviene that the hyroponi proeures use in this stuy are injurious to the plants. Uptake rates were alulate as esribe previously (Raab et al. 1996) base on linear rates of epletion utilizing a minimum of eight time points. Curvilinear behavior was only observe in some experiments at 80 90% epletion. Data from urvilinear patterns were not use in the rate alulations. All speies were examine for the presene of myorrhizae. One month after establishment, samples of roots were remove from two iniviuals of eah speies an either examine immeiately, or preserve in 90% formalin, 5% aeti ai, an 5% ethanol (FAA) for later inspetion. After staining with trypan blue (Mullen an Shmit 1993), the roots were examine uner a isseting mirosope. No olonization was observe for etomyorrhizae or arbusular myorrhizae. Soil properties Soil C an N ontents were measure along all four of the alpine ry meaow sites an at the shortgrass steppe. Repliate soil ores were han-augere to 15 m epth an sieve to 2 mm. Samples of mg were ombuste on a Europa Sientifi Traermass isotope-ratio mass spetrometer (Crewe, Unite Kingom) at the University of California, Berkeley. Due to the extremely low bulk ensity an high C ontent of the fen soil, it was not possible to obtain N measurements for this site by ombustion, as the tin apsules often olue the furnae inlet. Soil protease ativity was etermine at 3 4 wk intervals from May to Otober 1997 along the four alpine ry meaow transets. For the subalpine fen an shortgrass sites, omplete seasonal analyses for soil protease ativities were not onute. Rather, values were etermine uring the last week of August for the subalpine fen site, an the first week of September for the shortgrass steppe site. Amino ais were only observe uring the late summer months at these two sites (ata isusse later), an so the protease sampling sheme was esigne to reflet this observation. Repliate soil ores were obtaine from three points along the mirolysimetry transets an assaye within 24 h of olletion both with ( potential ) an without ( native ) protein amenment as asein. For the alpine soils, 2 3 g of soil were solubilize at a 1:5 (soil : buffer) ratio in 50 mmol/l Na-aetate buffer (ph 5.2, the mean ph of saturation pastes from the alpine sites) an 0.4 ml of toluene, an were inubate at 3 C for h. For the fen an shortgrass steppe soils, 50 mol/l potassium phosphate buffer (ph 7.5) was substitute in the assay to better math the ph of the fen an steppe soils. Ninhyrin analysis (Rosen 1957) of the supernatant after entrifugation allowe quantifiation of soluble -amino N release. Soil protein was etermine following a 1-h extration uner ontinuous shaking in 1 mol/l NaOH at a 1:5 (soil : solution) volumetri ratio. Following entrifugation at high spee, the supernatant was ilute 100-fol, an analyze for protein onentration using the metho of Brafor (1976). Soil moisture was etermine by time-omain refletometry (TDR) using a Tektronix (Beaverton, Oregon) 1502C metalli able tester. Signals from the three-ro, stainless steel TDR probes (ros m in iameter, 10 m in length) were propagate over 50 oaxial able. Transets of seven TDR probes at eah of the four alpine ry meaow sites were permanently installe in July The soil ieletri onstant was onverte to gravimetri water ontent using alibration urves obtaine from soil ores (Dalton an van Genuhten 1986). Measurements of soil moisture ontent were mae at monthly intervals throughout the 1996 growing season. Partitioning of NH 4, an the amino ais glyine an glutamate, between the soli an liqui soil phases was measure using an initial mass isotherm tehnique (Novin et al. 1986). Repliate ores of A-horizon soils from three of the four alpine transets were sreene to 2 mm, an 1-g subsamples autolave in 10 ml of a 10 mmol/l CaCl 2 solution (ph 5.5). Filtere (0.2 m) aliquots of various starting onentrations (0 1 mmol/l) of either NH 4 or amino ais ((L)-glutamate or glyine) were ae to eah soil/solution sample, an mixe on a reiproating shaker at 20 C for 2 h. Solis were pellete in a entrifuge at 5000 g, an the resulting supernatant quantifie for the ompoun of interest as esribe above. Soli liqui partitioning oeffiients (K, L/kg) were alulate as [10m/(1 m)], where m is the slope of urves from Fig. 8. Statistial analysis Signifiant ifferenes among means of uptake rates for ifferent speies were teste using Tukey s stuent-

6 2412 THEODORE K. RAAB ET AL. Eology, Vol. 80, No. 7 FIG. 1. Uptake rates for NH 4 (horizontally stripe bars), NO 3 (iagonally stripe bars), an glyine (soli bars) among 13 sege speies. Plants were ompare at an initial onentration of 125 mol/l for eah N form. Eah bar represents the mean 1 SE for n 3 5 iniviual plants, with values expresse per unit root ry mass. Speies esignation is aoring to the numbere sequene presente in Table 1. ize range test. Group ifferenes (i.e., ifferenes between the poole subalpine an alpine speies vs. the poole temperate an subtropial speies) were teste using ANOVA on mean values for eah speies. Differenes in protease ativity between alpine sites over all sampling ates were analyze by repeate measures ANOVA, using mean values for eah sampling ate. Minimum an maximum soil moisture ontents, an ifferenes among sites for total soil C or N were teste using ANOVA. Relationships between variables were teste using regression analysis. All statistial analyses were performe using SAS proeures, ANOVA, REG, an UNIVARIATE. RESULTS Measurements of N uptake by 13 speies of the Cyperaeae Most speies that were teste ha the apaity to take up all three forms of N: NO 3,NH 4, an glyine (Fig. 1). However, there were exeptions to this pattern. As note previously, the alpine sege Kobresia myosuroies laks the ability to take up NH 4 (Raab et al. 1996). This oes not appear to be a general feature of alpine seges, as both Carex rupestris an Carex albonigra oul take up NH 4. Nor is it a general feature of the genus Kobresia, as K. simpliiusula, a subalpine sege oul take up NH 4 at nearly the same rate as glyine. One of the 13 speies, the subtropial sege Gahnia gahniformis, i not take up glyine at measurable rates. There appeare to be a teneny for the three subalpine an two of the alpine speies to exhibit low rates of total N uptake, irrespetive of the type of N, ompare to speies from more temperate habitats in the shortgrass steppe, the swamp of North Carolina, an subtropial Hawai i (P 0.03). Exeptions to this pattern were foun for the two Kobresia speies, whih assimilate N at relatively high rates espite being native to subalpine an alpine habitats. (When isolate as a group, the two Kobresia speies exhibite total N uptake rates that were not ifferent from the temperate an subtropial speies [P 0.05].) As a group, the alpine an subalpine speies exhibite signifiantly higher rates of glyine uptake ompare to NO 3 an NH 4 (P 0.05). Caution shoul be use in interpreting the relative rankings of speies base on the experiments reporte here. It is possible that the rankings woul hange given measurements with ifferent onentrations of NH 4,NO 3, an glyine in the uptake uvette. Abunane an seasonal pattern of NO 3,NH 4, an amino ais in the soil-pore water of alpine, subalpine fen, an shortgrass steppe soils During the 1996 growing season, measurable amino ai onentrations were observe in the soil-pore water for at least one site among the four alpine ry mea-

7 Otober 1999 AMINO ACID AVAILABILITY TO SEDGES 2413 FIG. 2. Seasonal pattern of N ompoun onentration in the soil-pore water for NO 3 ( ), NH 4 ( ), an total amino ais ( ) for the (A) alpine ry meaow sites (the average observe for four ifferent transets), (B) the subalpine fen site, an (C) the shortgrass steppe site. Values with vertial bars represent mean 1 SE of 4 7 measurements. Values without vertial bars represent one observation. ows uring all months of the growing season (Fig. 2). Only uring the mile of the summer, however, were amino ai onentrations higher than those for NO 3 an NH 4. The only amino ais etete in the soilpore water at onentrations 10 mol/l (an arbitrary threshol for assessing the importane of amino ais to the N omposition of soil-pore water) were glyine an glutamate, with glyine generally twie as abunant as glutamate (ata not shown). At the subalpine fen, NO 3 was the ominant N speies in the soil-pore water early in the summer, with NH 4 ominating later in the summer. Amino ais were only observe late in the summer, an at onentrations that mathe those for NO 3 an NH 4. The only amino ai that was etete at this site in onentrations 10 mol/l was aspartate. At the shortgrass steppe site, NO 3 ominate the N omposition of soil-pore water at all but the earliest sampling ates. In general, NH 4 was low in onentration, or not etetable. Amino ais were etete only uring the latter portion of the growing season, being present at onentrations 25% those of NO 3. The only amino ai present in onentrations 10 mol/l was glutamate. The four alpine transets iffere in the pattern with whih amino ais were present in the soil-pore water aross the season (Fig. 3). At the E an W sites, amino ais were only observe early in the growing season. At the B site, amino ais were present early an late, but not uring the mile of the season. At the N site, amino ais were present uring the entire latter half

8 2414 THEODORE K. RAAB ET AL. Eology, Vol. 80, No. 7 (Fig. 5). (Total N ata was not available for the subalpine fen site.) Soils from all four of the alpine ry meaows exhibite higher protease ativities when supplemente with asein (P 0.05), although there were no ifferenes among sites with respet to native or potential (asein-amene) protease ativities (Fig. 6). The relationship between NaOH-extratable protein an protease ativity was linear when expresse aross sites from all three eosystems (Fig. 7). The subalpine fen an the shortgrass steppe soils ontaine lower amounts of protein an proportionately lower native protease ativity. Alpine soils from three of the ry meaow transets retaine glyine an glutamate to a higher egree than NH 4 (P 0.05) (Fig. 8). (Measurements were not mae for the fourth alpine transet.) Retention by these alpine soils range from 50 to 72% of the ae amino ai following a 2-h inubation. In ontrast, retention for NH 4 range from 30 to 45%. Retention of both amino ais an NH 4 was linearly epenent on the amount of ae ompoun. There were only slight FIG. 3. Seasonal pattern of total amino ai onentration in the soil-pore water of four ifferent alpine ry meaow transets. Values with vertial bars represent mean 1 SE of 2 5 measurements. Values without vertial bars represent one observation. of the season. The sites also iffere with respet to the onentration of amino ais in the soil-pore water that was ollete. At the N site, onentrations reahe a maximum of 158 mol/l, whereas onentrations at the W site never exeee 30 mol/l. Glyine ominate the amino ai spetrum of the alpine. Soil properties an soil protease ativities The four alpine ry meaows iffere with respet to soil moisture ynamis an the C an N ontent of the A soil horizon (Fig. 4). Site W ha a thinner A horizon than the other sites, with onomitantly lower soil moisture ontents. Site E exhibite the highest maximum an minimum soil moisture ontents of the four sites. Site W also exhibite lower C an N ontents in the A horizon relative to the other sites. The C:N ratio of the soils range from 8.5 for the N site to 14 for the W site. Within the four alpine ry meaows there was no signifiant orrelation between the NaOHextratable protein fration of the soil an the maximum or minimum soil moisture status or the total arbon ontent (P 0.05). When analyze for five of the stuy sites (the four alpine sites, an the shortgrass steppe site), soil NaOH-extratable protein onentrations were signifiantly orrelate with total N ontent FIG. 4. (A) Seasonal maximum (open bars) an minimum (soli bars) soil moisture ontents for the four alpine ry meaow transets. Vertial bars represent mean 1 SE of seven measurements along eah transet. (B) Total soil C (open bars) an N (soli bars) ontent for the four alpine ry meaow transets. Vertial bars represent mean 1 SE of 10 measurements along eah transet. Values marke with ifferent letters within a ategory are signifiantly ifferent at P 0.05.

9 Otober 1999 AMINO ACID AVAILABILITY TO SEDGES 2415 FIG. 5. NaOH-extratable protein in relation to total N for the four alpine ry meaow sites (esignate as W, E, N, an B) an the shortgrass steppe site (esignate as S). Data were not available for the subalpine fen site. Horizontal an vertial bars represent 1 SE for n 10. Data were ollete uring early July for the alpine sites an uring late August for the shortgrass steppe site, the approximate times of maximum soil protease ativities. FIG. 6. The ratio of native to asein-amene ativities of soil protease for the four alpine ry meaow sites. Values represent the seasonally average ativity 1 SE etermine from measurements at 3 4 wk intervals from May through Otober 1997 at three points along eah of the mirolysimetry transets. All ativities were etermine at 3 C. Only at the W site was the ratio marginally signifiant (P 0.08) in omparison to the other sites. an nonsignifiant (P 0.05) ifferenes in the retention patterns for soils from the ifferent ry meaow sites. DISCUSSION Amino ai uptake by the roots of Cyperaeae speies Several past stuies have emonstrate that plants in ertain eosystems have the apaity to utilize organi ompouns as a nitrogen soure (e.g., Abuzinaah an Rea 1986, Bajwa an Rea 1986, Chapin et al. 1993, Kiellan 1994, Raab et al. 1996, Shmit an Stewart 1997, Nasholm et al. 1998). To ate, these stuies have fouse on four eosystems: eriaeous heath, the arti tunra, boreal forest, an the alpine. All four of these eosystems are haraterize by soils high in organi matter, with strong seasonal onstraints on mirobial mineralization. In the ase of eriaeous heath an boreal forest, myorrhizal assoiations between plants an fungi also ontribute to a tight symbiosis whereby plant utilization of organi N is failitate by fungal uptake (Rea 1993). Other than a general reognition of the importane of organi N in these eosystems, little is known about this moe of nutrition in other eosystems, the soil proesses that promote organi N availability, an the apaity for retention of organi N in ifferent soils. The results of the urrent stuy emonstrate that within a phylogenetially restrite group of plants the apaity for amino ai uptake is wiesprea but highly variable. Of the 13 speies of Cyperaeae that were assaye, only one lake the ability to take up glyine. Kiellan (1994, 1997) observe amino ai uptake in nine ifferent arti tunra speies. In the urrent stuy, myorrhizal fungi were absent in all of the stuie speies. The wiesprea nature of glyine uptake among the speies surveye in this stuy, an those surveye in the Kiellan (1994) stuy, suggests that the limiting fator to organi N uptake may not rest in the inability of roots to absorb organi ompouns. Rather, the limiting fator is likely to resie in the availability of amino ais in the soil, whih in turn is ontrolle either by rates of proteolysis or the exhange of amino ais between the soli an liqui phases. The fat that we ientifie one sege speies as being inapable of absorbing glyine, an that there was FIG. 7. Soil protease ativity in relation to NaOH-extratable protein for six ifferent sites (the four alpine ry meaow transets represente as N, B, E, an W; the subalpine fen represente as F; an the shortgrass steppe represente as S). Horizontal an vertial bars represent 1 SE for n 10. In orer to stanarize for temperature effets on protease ativity, all assays were onute at 3 C. Measurements were mae on samples ollete the same ay for all sites in early July for the four alpine sites, an in late summer for the subalpine fen an shortgrass steppe sites. Thus, the measurements were mae near the time of seasonal maximum protease ativity for all sites.

10 2416 THEODORE K. RAAB ET AL. Eology, Vol. 80, No. 7 exhibite high rates of N uptake, espeially with respet to glyine. The physiologial reason for these high uptake rates is unknown. The fat that these high rates seem to stan out ompare to other eologially similar speies, an that they our in ongeners, suggests that they are ue to seletion in some past environment with retention ue to phylogeneti inertia. When onsiere as a group, the alpine an subalpine speies exhibite higher rates of glyine uptake relative to NO 3 or NH 4 uptake. This may reflet evolution towar speialization on the uptake of organi N, relative to inorgani N in these speies. FIG. 8. N-retention isotherms for soil from three of the four alpine ry meaow sites. The amount of initial N that was ae an that is retaine or release from the inubate soil is represente on the y-axis. The slopes for glyine an glutamate are not signifiantly ifferent from eah other (P 0.05) but are signifiantly ifferent from that for NH 4 (P 0.05). broa variation in glyine uptake rates among speies, iniates that the apaity to utilize glyine is uner geneti ontrol an is potentially subjet to seletion in ifferent eologial onitions. In this stuy, we were able to isern two eologial trens in the istribution an magnitue of the trait. In general, the subalpine an alpine speies exhibite lower rates of glyine uptake, ompare to the speies from more temperate habitats. This appeare to be a onsequene of higher N uptake rates in general in the temperate speies, sine the rates of NO 3 an NH 4 uptake were also higher. It is likely that the temperate speies exhibit higher growth rates than the alpine an subalpine speies (though this was not quantitatively measure). If this is the ase, then the higher rates of glyine uptake in temperate speies may be more refletive of evolutionary influenes on plant growth pattern than evolutionary response to unique features of the organi N ontent of temperate soils ompare to subalpine an alpine soils. The one exeption to the low rates of N uptake in subalpine an alpine speies ourre in the genus Kobresia. Both the alpine an subalpine Kobresia speies Soil proesses an pool sizes of amino ais Of the three ifferent eosystems that were stuie, amino ais were most abunant in the alpine soils, being present at measurable quantities at all times uring the growing season (Fig. 2). Among the four alpine ry meaows there was onsierable variability in the amount an timing of amino ai availability (Fig. 3). Although these results oul be use to suggest highly stohasti patterns in the availability of amino ais, we woul avoate aution. Measurements using mirolysimeters are somewhat onservative in their estimate of amino ai availability. This is beause soilpore water has to be relatively abunant for the mirolysimeter to yiel a measurable sample. It is likely that plants an generate a matri potential low enough to extrat nutrients at soil moisture ontents that woul not yiel a measurable mirolysimeter sample. When all four alpine meaows are onsiere, there were 40 mirolysimeters available for sampling. During the growing season, these mirolysimeters were heke weekly over a 14-wk perio for the presene of measurable sample. Of the approximately 600 heks that were mae on these mirolysimeters, only 32 olletions were mae uring 1996, with 21 yieling etetable amino ais. Thus, the presene of amino ais in the mirolysimeter samples shoul be taken as eviene that the soil-pore water of alpine ry meaows frequently ontains amino ais, but the preise temporal pattern of availability annot be iserne. When ombine with the observations that alpine seges are apable of absorbing amino ais, the mirolysimetry results support the onlusion that amino ai N is inee an important omponent of the alpine sege N buget. To plae the possible use of amino ai N into the broaer ontext of the alpine N yle, one an alulate the rate of proteolysis require to satisfy the annual N requirement of Kobresia myosuroies. The annual N requirement for K. myosuroies in the ry meaow habitat of Niwot Rige, Colorao is 3.3 g N/m 2 (Fisk et al. 1998). Using our measurements of the soil bulk ensity (0.5 g/m 3 ), assuming Q 10 2 for soil proteolysis an an effetive rooting epth of 10 m, an saling measure alpine proteolysis rates (unamene, at 3 C) to a seasonal sinusoial temperature regime

11 Otober 1999 AMINO ACID AVAILABILITY TO SEDGES 2417 with a snow-free season of 100, we estimate annual turnover of protein N to be 42 g N/m 2. Thus, K. myosuroies oul obtain all of its require N from 5 6% of annual proteolysis. (Muh of the N release from proteolysis has been shown to be immobilize by summer rhizosphere baterial populations [Lipson an Monson 1998].) At the subalpine fen an shortgrass steppe sites, amino ais were only present in soil-pore water late in the growing season. At these sites, amino ai availability was never observe to be as high as that for the alpine soils. Furthermore, unlike alpine soils, glyine was not the most abunant amino ai. Past measurements have shown that non-myorrhizal roots of some seges an take up glutamate, in aition to glyine, but at onsierably slower rates (Kiellan 1994, Raab et al. 1996). Thus, seges at the shortgrass steppe site may be apable of utilizing glutamate as a soure of N late in the growing season, though perhaps not as effetively as the ase for glyine uptake by alpine plants. Measurements of uptake rates for aspartate, the most abunant amino ai at the subalpine fen site, have not been onute for our sege speies, though Kiellan (1994) observe signifiant aspartate uptake in exise roots of Carex aquatilis an Eriophorum vaginatum. Kiellan (1995) then onsiere amino ai onentrations in water extrats of four ifferent Alaskan eosystems, an observe serine, glyine, aspartate, glutamate, an arginine to aount for 50% of the free amino ai pool. Vermuolls of the Jura Mountains extrate with K 2 SO 4 likewise possesse alanine, glyine, an glutamate as major omponents of the rapi-turnover N fration (Lemaitre et al. 1995). The abunane of soil protein at the six stuy sites explaine muh of the variation in protease ativity. Soil protein ontent was also highly orrelate with total N ontent. Thus, total N ontent an onomitantly protein availability, appear to limit amino ai generation through soil proteolysis. This is supporte by the fat that protein supplementation stimulate soil protease ativities at all stuy sites. The fat that neither native nor asein-amene rates varie among alpine sites suggests that other fators influene expression of this ativity in situ. Aitional fators that may influene protease ativities are ph, metal availability, an soil temperature. One free from the protein fration, amino ais appear apable of relatively high retention on the alpine soil matrix (Fig. 8). An inex of the tenaity with whih nutrients are retaine in the soil is the soli liqui partition oeffiient (the so-alle K value) whih is erive from the slope of the asorption isotherms presente in Fig. 8. With respet to NH 4 retention, K values have been measure as 4 L/kg for volani soils in New Zealan (Theng 1972) an L/kg for flooe rie soils in the southeastern Unite States (Rao et al. 1984). These values are interprete to iniate that NH 4 is tightly boun to the soli matrix, an onsierably less available for plant uptake in the volani soils, with higher availability in the flooe rie fiel soils. The alulate K for NH 4 in the alpine soils of this stuy range from 4 to 17 L/kg among three of the four alpine ry meaows that were stuie, iniating strong retention of NH 4. The alulate K values for amino ais range from 12 to 24 L/kg for glyine an 12 to 21 L/kg for glutamate. The only other stuies of whih we are aware that have erive the K for amino ais have alulate a value of 6 L/kg for glyine retention on the lay mineral montmorillonite (Greenlan et al. 1965), an 2.9 L/kg for glyine retention on a spoi B-horizon (Jones et al. 1994). When taken together, the K inies for NH 4 an amino ais in the alpine ry meaow soils reflet an important role for the soil in asorbing an retaining nitrogen ompouns. Past stuies using 15 NH 4 have also emonstrate that alpine ry meaow soils represent strong ompetitive sinks for inorgani N. Jaeger et al. (1998) foun that uring June when soils were still relatively moist, 15 NH 4 that was ae to the soil was reaily assimilate by plants an miroorganisms (83% of the label was reovere in these frations). However, by early August, when the soils were rier an plants ha reahe their maximum seasonal biomass, most of the ae 15 NH 4 label was retaine by the soil an was not available for uptake by plants or miroorganisms (35% of the label was reovere in the plant an mirobial frations). In a seon stuy, Lipson an Monson (1998) ae ual-labele glyine ( 13 C an 15 N) to miroosms ontaining alpine ry meaow soils from the same site use in the urrent stuy, an reovere 43% of the 15 N label in the bulk soil. Suh high retention may be ommon for soils with high organi ontent. Sposito (1989) has onlue that in soils with organi matter ontent 8%, inreasing organi matter ontrols asorption an retention of ations ompare with the lay fration. The nature of the interation between organi matter an ion retention is not well unerstoo, but sorptive preservation of amino ais (sensu Keil et al. 1994) may effetively eouple their proution from eventual release to plants. In all of the stuies isusse here, extration of amino ais from the soil was aomplishe with water. While this approah is most realisti with respet to those proesses that ontrol the appearane of amino ais in soil-pore water, it may be onservative with respet to the ability of plant roots to extrat amino ais from the soil. The role of the soil as both a sink for the uptake of organi N an a reservoir from whih organi N may be aesse by plants shoul beome more lear through the use of measure K values an soil protease ativities to onstrain soil nutrient transport moels as they are use to assess the balane between soil amino ai generation an uptake to the plant, mirobe, an soil frations. This type of analysis is urrently unerway in our laboratory.

12 2418 THEODORE K. RAAB ET AL. Eology, Vol. 80, No. 7 Conlusions The results of this stuy support the hypothesis that the potential to take up soil amino ais by non-myorrhizal roots is wiesprea in the Cyperaeae. Plants of this family are apable of absorbing amino ais in habitats other than the alpine an arti, the most wellstuie situations, though ompare to alpine eosystems, the opportunities to utilize soil amino ais in the subalpine fen an shortgrass steppe eosystems appear limite. In the alpine eosystem, where we have obtaine the most omplete set of ata, the soils possess relatively high apaities to generate amino ais through protease ativity, whih appears to be linke to the high N, an onomitant high protein ontents, in these soils. The retention of soil amino ais may inrease with elevate organi matter levels, leaing to the onlusion that the soil funtions in a ual role as both a reservoir for amino ais to be aesse by plant roots, an as an effetive apaitor eoupling proution of amino ais from their eventual release to plants an soil miroorganisms. ACKNOWLEDGMENTS The authors are grateful for the omments provie by Dr. Steve Shmit an two anonymous reviewers on an earlier raft of the manusript. The authors woul like to thank Dr. Davi Foote, U.S. Park Servie, for permission to ollet plants within Hawai i Volanoes National Park, an Ruth Ley for assistane in site seletion an plant propagation. Dr. Alan T. Carpenter of the Nature Conservany s Colorao Chapter graiously provie aess to High Creek Fen Reserve for researh. Dr. William Lauenroth provie aess to the CPER researh enlosure. The USDA Western Regional Plant Introution Servie generously provie sees. We thank Chris Shat for mirosopi examination of several root samples for myorrhizae. This work was supporte by NSF grant to R. K. Monson. Logistial support of the University of Colorao Mountain Researh Station through the NSF-fune LTER program (DEB an BIR ) is greatly appreiate. LITERATURE CITED Abuzinaah, R. A., an D. J. Rea The role of proteins in the nitrogen nutrition of etomyorrhizal plants. III. Protein utilization by Betula, Piea an Pinus in myorrhizal assoiation with Hebeloma rustiliforme. New Phytologist 103: Bajwa, R., an D. J. Rea Utilization of mineral an amino-nitrogen soures by the erioi myorrhizal enophyte Hymenosyphus eriae an by myorrhizal an nonmyorrhizal seelings of Vainium. Transations of the British Myologial Soiety 87: Birkelan, P. W., R. M. Burke, an R. R. Shroba Holoene alpine soils in gneissi irque eposits, Colorao Front Range. Pages 1 21 in J. W. Haren, eitor. Soil hronosequenes in the western Unite States. U.S. Geologial Survey Bulletin Brafor, M. 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