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1、EnvironmentalScienceandPollutionResearchhttpsdoi.org10.17sll356-021-16817-8RESEARCHARTICLEDeterminantsofsoilcarbon-andnitrogen-hydrolyzingenzymeswithindiferentaforestedlandsincentralChinaQianxiLi国 Xiaoli Chengxlcheng Hubei Provincial Academy of Eco-environmental Sciences (Hubei Eco-environmental Eng
2、ineering Assessment Center), Wuhan 430079, Peoples Republic of China* Schl of Environmental Studies. China University of Geosciences, Wuhan 430074, Peoples Republic of ChinaYiranDong2QianZhang, Key Laboratorjf of Soil Ecology and Health in Universities OfYunnan Province. School of Ecology and Enviro
3、nmental Science, Yunnan University, Kunming 650091, Peoples Republic of ChinaWeiJia Key Laboratory of Aquatic Botany and Watershed Ecology. Wuhan Botanical Garden, Chinese Academy of Sciences (CAS), Wuhan 430074, Peoples Republic of ChinaXiaoIiCheng3Received:27May2021/Accepted:25September2021TheAuth
4、or(s),underedusivelicencetoSpringer-VerlagGmbHGermany,partofSpringerNature2021AbstractSoilorganicmatter(SOM)decompositionisregulatedbyacomplexsetofenzymes.However,theinfuencesofbioticandabioticfactorsonspatialvariationsofsoilenzymeactivity(EA)withinecosystemsremainunresolved.Here,wemeasuredEAatdifer
5、entlocationswithintwoaforestedlands(coniferouswoodlandandleguminousshrubland),andsimultaneouslycollecteddataonsoilphysico-chemical,vegetation-related,andmicrobialpropertiestoidentifythedeterminantsofEAspatialpatterns.TheresultsshowedthatsoilorganicCandtotalNcontentswerethepredominantabioticfactorsin
6、regulatingabsoluteEA(EAperunitofoven-drysoilmass)inbothaforestedlands,whilesoilpHwasthepredominantfactorinregulatingspecifcEA(EAperunitofmicrobialbiomass(MB).However,thepredominantbioticfactorsvariedwiththeaforestedtype:therootbiomassandMBwerethedeterminantsofEAintheshrubland,whereasthetreedistribut
7、ion,litterandrootbiomass,andbacterialbiomasswerethedeterminantsinthewoodland.Vegetation-relatedfactors(i.e.,litterandrootbiomass)indirectlyinfuencedsoilEAbyregulatingthesoilabioticfactors.ComparedwiththeMB,microbialcommunitycompositionhadaminorimpactonEA.ThevarianceofspecifcEA(EAperunitofMBorSOM)exp
8、lainedbyselectedfactorswasmuchlowerthanthatofabsoluteEA.Inaddition,theenzymaticC/Nratiowithinecosystemsdidnotfollowageneralpattern(1:1)observedataglobalscale.Ourresultsprovidenovelexperimentalinsightintoecosystem-levelspatialvariabilityofCandNcyclingviaenzymes,suggestingthatsoilabioticfactorsaremore
9、reliablethanbioticfactorstorefectEAspatialpatternsacrossaforestedsystems.KeywordsSoilenzymeactivitySpatialvariationAforestationEnzymestoichiometryVariationpartitioningpublishedonline:26october2021ResponsibleEditor:RobertDuranIntroductionGiventhatsoilscontainthelargestreservoiroforganiccarbon(C)andni
10、trogen(N)inthebiosphere(Lal2004;LehmannandKleber2015),soilorganicmatter(SOM)mineralizationcouldpotentiallyregulateglobalCandNcycling.Meanwhile,soilenzymesareregardedasproximateagentsofSOMmineralizationbecausetheyreducetheactivationenergyofrate-limitingreactionsandspeedupthebreakdownofpolymericmacrom
11、oleculesintolowmolecules(Nannipierietal.2012;Bumsetal.2013;Maoetal.2015).Therefore,accuratepredictionofsoilenzymeactivity(EA)canprovideusefulinformationaboutSOMturnoveraswellasitsresponsetoanthropogenicdisturbancesandenvironmentalchanges(Hanetal.2019).However,thepredictionofsoilEAiscomplicatedsincee
12、nzymesarenotevenlydistributedintheenvironment(Baldrian2014).RelativelyhighspatialvariabilityofsoilEAhasbeenobservedevenwithinasinglefeldbecausesoilEAareregulatedbymultiplebioticfactors(e.g.,vegetationandmicrobialcommunitycomposition)andabioticfactors(e.g.,soilphysico-chemicalpropertiesandmicroclimat
13、e).Inrecentdecades,anumberofstudieshavebeenconductedtoexplorethespatialvariationsofsoilEAandtheirdeterminantsataglobalorregionalscale.Basedonthesestudies,soilpHandSOMcontentwereidentifedasthemostimportantabioticfactorsafectingsoilEA,especiallyforsoilhydrolaseactivity(Sinsabaughetal.2008;KivlinandTre
14、seder2014;Xuetal.2020).However,thespatialvariabilityofsoilEAwithinecosystemshasbeenlargelyneglected(Banerjeeetal.2016;Stursovaetal.2016).Whetherobse,ationsatlargescalescouldapplytoasingleecosystemisstillanopenquestion.AlthoughafewstudieshaveinvestigatedthespatialheterogeneityofsoilEAwithinecosystems
15、andthedeterminants,theseconclusionsareindebate.Forexample,Boeddinghausetal.(2015)reportedthatsoilpHwasanimportantdeterminantofspatialdistributionofEAinagrasslandecosystem,whilestudiesconductedinforestecosystemsandinagrassland-woodlandecotonefoundthatsoilpHwasnotspatiallyconnectedwithsoilEAbecauseoft
16、heverylowvariationsofpHobservedinthesestudies(Banerjeeetal.2016;tursovaetal.2016).Mayoretal.(2016)investigatedthespatialdiferencesinsoilEAlevelsbetweenvegetationpatchesandinter-patchesinashrublandecosystem,andfoundthatvegetationcanopycouldafectsoilEApatternsthroughrhizosphereefectsandsubstrateinputb
17、yIitterfall(Dornbush2007;Brzosteketal.2013;Fengetal.2019).Bycontrast,vegetationpropertiesdidnotSignifcantlyinfuencesoilEAinaforestecosystem(Slursovdetal.2016).Meanwhile,soilmicrobialbiomass(MB)andcommunitycompositionhavebeensuggestedasdirectregulatorsofsoilEAspatialpatternssincetheproductionofaspeci
18、fcenzymeisinducedbysomeparticularspecies(BaldrianandSnajdr2011;Bowlesetal.2014),butexceptionswerealsofoundinotherstudiespartlydueIothatalargeportionofsoilMBwasmetabolicallyinactive(Bocddinghausetal.2015;Stursovdetal.2016).Inaddition,soilpHwasfoundtobecorrelatedwithCellobiohydrolaseandchitinaserather
19、than-and-glucosidasesinagrasslandecosystem.AllthesecontradictoryresultsindicatedthattheefectivenessofbioticandabioticvariablesinregulatingsoilEAwithinecosystemswouldvarydependingontheecosystemtypeorenzymetype.Despiteofthesestudies,onlyfewstudiescomparedtheefectsofsoilphysico-chemicalvariables,vegeta
20、tionproperties,andmicrobialcommunitytogetheronthevariationsofsoilEAwithindiferentecosystems.Moreover,asmultiplefactorsareinterrelated(Walleniusetal.2011),therelativecontributionsofthesefactorstosoilEAspatialpatternshavenotbeenwellinvestigated.SoilEAcanbeexpressedindiferentformsincludingtheabsoluteac
21、tivity(i.e.,activityperunitofoven-drysoilmass)andthespecifcenzymeactivity(i.e.,activityperunitofSOMorMB).ThespecifcEAcanbeusedtoeliminatetheimpactofSOMorMB,andtestwhethervariationsofsoilEAcanoccurindependentlyofvariationsofSOMorMB(RaiesiandBeheshti2014).Todate,muchlessisknownabouthowthespecifcEAresp
22、ondspatiallytootherabioticandbioticfactorsatsmallscaleswithindiferentecosystems.Inaddition,therelativeabundanceofsoilC-andN-hydrolyzingenzymes(i.e.,enzymaticC/Nratio),namely,enzymestoichiometry,exhibitsthepotentialtorefectthebiogeochemicalequilibriumbetweenmicrobialCandNdemandsandnutrientavailabilit
23、yoftheenvironment(Sin-sabaughetal.2009;Mooshammeretal.2014).Thus,spatialvariationsofenzymaticC/NratiowithinecosystemscouldprovideafunctionalassessmentoftherelativeresourcelimitationsofmicrobialmetabolismandtherelativeratesofSOMdecomposition(Sinsabaughetal.2009;Liaoetal.2021).Previousstudiesconcernin
24、genzymestoichiometrypatternshavebeenmainlyconductedatalargeorglobalscale(Sinsabaughetal.2008;PengandWang2016).ThesestudieshaveshownthatratiosofC-andN-hydrolyzingenzymeactivitiesconvergedon1:1.Itislessclearifthepatternofenzymestoichiometryataglobalscalecanbeappliedwithinecosystems.Inthepresentstudy,w
25、einvestigatedspatialvariationsofsoilC-andN-hydrolyzingenzymes(defnedasenzymesthatcontributetothehydrolysisoforganicCandNcompoundsinsoils)withintwotypicalaforestedlands(woodlandandshrubland),aswellasthefactorsthatbestregulatingthesevariations.WehypothesizedthatthespatialvariationsofEAwithinecosystems
26、couldbeexplainedbythecombinationoffactorsrelatedtosoilenvironment,vegetation,andmicrobialcommunity,butthemajordeterminantsofthesevariationswouldVarydependingontheecosystemtypeandenzymetype.Inaddition,toexploretheallocationofC-andN-hydrolyzingenzymes,wealsohypothesizedthattheenzymaticC/Nratiowithinec
27、osystemsshouldbesimilartothatataglobalscale,whichconvergedon1:1.MaterialsandmethodsStudysiteandsamplingdesignThestudywasconductedintheexperimentalareaoftheWulongchiResearchStation(32o45,N,111o13,E),HubeiProvince,China.Primaryforestsinthisregionwereconvertedtocroplandsabout70yearsagoasaresultoftheres
28、ettlementofinhabitants(LiandZhang2008).Largeareasofcroplandswerelaterconvertedtoopenareaswithnovegetationcoverduetointensivelanddegradation.Sincethe1980s,aforestationhavebeenimplementedinthisregion(Zhuetal.2010).Asaresult,mostoftheopenareashavebeenconvertedtowoodlandandshrublandplantations.Thetwoafo
29、restedlandswereunderdifferentnutrientregimes,namely,thatthewoodlandsitewasplantedwithconiferoustrees(Platycladusorientalis(Linn.)Franco,characterizedbyhighC/Nratio),whiletheshrublandsitewasplantedwithleguminousN-fxingshrubs(Sophoradavidii(Franch.)Skeels).Thisdiferenceinlitterqualitybetweenthetwosyst
30、emsresultedindifer-entnutrientregimesanddiferencesinsoilenvironmentandmicrobialcommunity(Table1).Managementsuchasfertilizationandirrigationontheseareashasbeenminimal.BothoftheaforestedsystemsthatdominatedbythetwospeciesalsowidelydistributeinnorthernChina.AstandofIO10mconsistingof22mgridcellswaslaido
31、utineachofthesitesincludingwoodland,shrubland,andtheopenarea(i.e.,control)inApril2017.Surface(0-10cm)soilsamplewascollectedusingacoreaugerateachnodeofthesegrids,resultingin36samplesperstand(Fig.1).Beforesoilsampling,wecompletelycollectedtheabovegroundlitterusinga0.20.2mframeateachnode.Alllivingroots
32、ineachsoilsampleswerecarefullyseparatedfromdeadrootsandwashed.Litlerandlivingrootswereovendriedat65toaconstantweighttoobtainthelitterandlivingrootbiomass(gm2).Allplantsthatlocatedinsideandaroundthe100-m2standwithdiameteratbreastheight(DBH)1cmweremeasured,andtheirgeographiccoordinateswererecorded(Fig
33、.1).SoilanalysisEachfreshsoilsamplewassievedwitha2-mmmesh.Aportionofeachsoilsamplewasfreezedriedforthemeasurementofphospholipidfattyacids(PLFAs),andanotherportionofsoilsampleswerestoredat4forthedeterminationofsoilEAwithin72h.Theremainingsoilswereairdriedforthedeterminationofothersoilproperties.Soilm
34、oisturecontent(SMC)wasobtainedgravimetricallybyOven-drying2()goffreshsoilat105toconstantweight.SoilpHwasmeasuredaftershakingasoil-watersuspension(1:2.5)for30minwithadigitalpHmeter.Soilorganiccarbon(SOC)andtotalnitrogen(STN)concentrationsweredeterminedonanelementalanalyzer(ThermoScientifcFlash2000HT,
35、Germany)afterremovinginorganicmatterbytreatingwith1MHCl(Chengetal.2013).ParameterEcosystem typeWoodlandShrublandCV (%)Open areaMeanCV (%)MeanMeanCV (%)Local environmentsLitter biomass (g m2)823.9a55161.7b94Root biomass (g m 2)560a58139.2b143Soil pH8.1c28.3b38.8a1SMC (%)19.3a2110.8b354.3c22SoilON19.4
36、a2811.5b139.4c16SOC (g kg ,)21.04a416.24b851.29c18STN (gkg-)1.12a380.54b790.14c21RIC (gkg ,)14.99a444.47b651.20c33RIN (g kg l)0.37a410.14b710.10b23Microbial PLFA biomass (nmol g 1 dry soil)Bacterial PLFA74.59a5411.15b656.64c70Fungal PLFA12.33a602.23b731.41c79G* PLFA23.64a573.88b80l.I7c62G PLFA50.57a
37、557.27b595.48c73ACT PLFA14.69a621.73b810.52c65AMF PLFA5.17a620.73b790.19c67Table 1 Mean and coefcients of variation for vegetation, soil, and microbial parameters within each site in the study areaLowercase letters indicate diferences among land types based on an ANOVA with post hoc comparisonsSMC,
38、soil moisture content; SOC, soil organic carbon; STN, soil total nitrogen; RIC, recalcitrance index for C; RIN, recalcitrance index for N: F. total fungi: B. total bacteria; G*, gram-positive bacteria; G-, gram-negative bacteria; AMF, arbuscular mycorrhizal fungi; ACT, actinobacteriaSoilrecalcitrant
39、C(RC)andN(RN)concentrationswereobtainedbyacidhydrolysis(seemoredetailsinXuetal.2015).Briefy,500mgofair-driedsamplewastreatedwith25mlof2.5MH2SO4.Theresiduewasrecoveredbyrepeatedcentrifugation,andthentreatedwith2mlof13MH2SO4overnight.Theremainingresiduewasrecoveredagainasdescribedaboveandthendriedat60
40、Eastin(mlFig.1SoilsamplingschemeandtreedistributioninthethreekindsofecosystemsintheDanjiangkouReservoirarea.Blueasterisksrepresentsoilsamplingsites.RedcirclesintheWoodlandrepresentPlat-ycladusorientalis(Linn.)Franco.RedandgraycirclesintheshrublandrepresentSophoradavidii(Franch.)SkeelsandViiexnegundo
41、L.var.Cannabifolia(SiebetZucc.)Hand.-Mazz.respectively.Thesizeofthecircleinthewoodlandandshrublandisproportionaltotreediameteratbreastheightundshrubbasaldiameter,respectivelyformeasuringRCandRNontheelementalanalyzer.TherecalcitranceindicesforCandN(RICandRIN,respectively)werecalculatedastheratiosofRC
42、andRNtototalCandN.ThemicrobialbiomassandcommunitystructurewereassessedbyusingPLFAanalysis(BossioandScow1998)(seemoredetailsinWuetal.2017).SpecifcPLFAmarkerswereusedtoquantifydiversetaxonomicgroups,includinggram-positivebacteria(G)gram-negativebacteria(G-),actinomycetes(ACT),arbuscularmycorrhizalfung
43、i(AMF),totalfungi(F),andtotalbacteria(B)accordingtoXuetal.(2015)andareshowninTableSl.Amodifedmethodbasedonfuorescent-linkedsubstratemicro-plateprotocolwasusedtomeasuretheactivitiesofenzymes(including-glucosidase(BG),/V-acetyl-glucosaminidase(NAG),andleucineaminopeptidase(LAP)insitupHconditionsandtem
44、perature(Smithelal.2016).Inthepresentstudy,weselectedthesesoilC-andN-hydrolyzingenzymesduetothefollowingreasons:(1)hydrolysishasbeenconsideredmoreimportantthanoxidationinSOMmineralizationprocesses(Nannipierietal.2012),(2)theiractivitiesareusuallymuchhigherthanotherhydrolasesacrossdiferentecosystems(
45、Bowlesetal.2014;Schimeletal.,2017;Zhangetal.2019),and(3)theratioofthethreeenzymes(InBG:ln(NAG+LAP)wasgenerallyusedtorepresentenzymestoichiometry(Sinsabaughetal.2008;PengandWang2016).Inbrief,1gofsoilwashomogeneouslydispersedin90mlofsodiumacetatebufer.ThepHofthebuferwasadjustedto8.0.Thepreparedsoilsus
46、pension,standardsolution,andfuorescentsubstratesolution(200Mintotal)weresuccessivelyaddedintoa96-wellmicroplate(WhatmanInc.,FlorhamPark,NJ).Thecompounds7-amino-4-methyIcoumarin(MUC)and4-methylumbclliferone(MUB)wereusedasthestandardreferencesforLAPandIheremainingenzymes,respectively.Themicroplatewasi
47、ncubatedat25for3.0h,andthen50lof1MNaOHwasaddedtostopthereaction.Thereleasedfuorescencewasdeterminedusingamultifunctionalmicroplatereader(TecanInfniteM200pro,Salzburg,Austria).Thewavelengthofexcitationusedwas360nmandtheemissionusedwas450nm.UnitsforabsoluteEAwereexpressedasnanomolesoffuorescenepergram
48、ofdryfractionperhour.EAwerenormalizedbyMBandSOMtoobtaindifferentspecifcEA(EA:MBandEA:SOM).CalculationandstatisticsWeusedaproximityindex(treeinfuencepotential,IP)toquantifytheinfuenceoftreedistributiononsoilEA(SaetreandB熊th2000).ThisindexrefectsthecombinedinfuenceofneighboringtreesonsoilEAateachsamplingpoint,andisexpressedusingEq.(1):P=iDBHiep(-cdi)(1)whereDBHiisthediameteratbreastheightorbasaldiameterofplantiinmeters,CiSascalingcoefficientandwassett
