Determinantsofsoilcarbonandnitrogenhydrolyzingenzymes.docx

EnvironmentalScienceandPollutionResearchhttpsdoi.org10.17sll356-021-16817-8RESEARCHARTICLEDeterminantsofsoilcarbon-andnitrogen-hydrolyzingenzymeswithindiferentaforestedlandsincentralChinaQianxiLi国 Xiaoli Chengxlcheng Hubei Provincial Academy of Eco-environmental Sciences (Hubei Eco-environmental Engineering Assessment Center), Wuhan 430079, People's Republic of China* Schl of Environmental Studies. China University of Geosciences, Wuhan 430074, People's Republic of ChinaYiranDong2QianZhang', Key Laboratorjf of Soil Ecology and Health in Universities OfYunnan Province. School of Ecology and Environmental Science, Yunnan University, Kunming 650091, People's Republic of ChinaWeiJia Key Laboratory of Aquatic Botany and Watershed Ecology. Wuhan Botanical Garden, Chinese Academy of Sciences (CAS), Wuhan 430074, People's Republic of ChinaXiaoIiCheng3Received:27May2021/Accepted:25September2021©TheAuthor(s),undere×dusivelicencetoSpringer-VerlagGmbHGermany,partofSpringerNature2021AbstractSoilorganicmatter(SOM)decompositionisregulatedbyacomplexsetofenzymes.However,theinfuencesofbioticandabioticfactorsonspatialvariationsofsoilenzymeactivity(EA)withinecosystemsremainunresolved.Here,wemeasuredEAatdiferentlocationswithintwoaforestedlands(coniferouswoodlandandleguminousshrubland),andsimultaneouslycollecteddataonsoilphysico-chemical,vegetation-related,andmicrobialpropertiestoidentifythedeterminantsofEAspatialpatterns.TheresultsshowedthatsoilorganicCandtotalNcontentswerethepredominantabioticfactorsinregulatingabsoluteEA(EAperunitofoven-drysoilmass)inbothaforestedlands,whilesoilpHwasthepredominantfactorinregulatingspecifcEA(EAperunitofmicrobialbiomass(MB).However,thepredominantbioticfactorsvariedwiththeaforestedtype:therootbiomassandMBwerethedeterminantsofEAintheshrubland,whereasthetreedistribution,litterandrootbiomass,andbacterialbiomasswerethedeterminantsinthewoodland.Vegetation-relatedfactors(i.e.,litterandrootbiomass)indirectlyinfuencedsoilEAbyregulatingthesoilabioticfactors.ComparedwiththeMB,microbialcommunitycompositionhadaminorimpactonEA.ThevarianceofspecifcEA(EAperunitofMBorSOM)explainedbyselectedfactorswasmuchlowerthanthatofabsoluteEA.Inaddition,theenzymaticC/Nratiowithinecosystemsdidnotfollowageneralpattern(1:1)observedataglobalscale.Ourresultsprovidenovelexperimentalinsightintoecosystem-levelspatialvariabilityofCandNcyclingviaenzymes,suggestingthatsoilabioticfactorsaremorereliablethanbioticfactorstorefectEAspatialpatternsacrossaforestedsystems.KeywordsSoilenzymeactivitySpatialvariationAforestationEnzymestoichiometryVariationpartitioningpublishedonline:26october2021ResponsibleEditor:RobertDuranIntroductionGiventhatsoilscontainthelargestreservoiroforganiccarbon(C)andnitrogen(N)inthebiosphere(Lal2004;LehmannandKleber2015),soilorganicmatter(SOM)mineralizationcouldpotentiallyregulateglobalCandNcycling.Meanwhile,soilenzymesareregardedasproximateagentsofSOMmineralizationbecausetheyreducetheactivationenergyofrate-limitingreactionsandspeedupthebreakdownofpolymericmacromoleculesintolowmolecules(Nannipierietal.2012;Bumsetal.2013;Maoetal.2015).Therefore,accuratepredictionofsoilenzymeactivity(EA)canprovideusefulinformationaboutSOMturnoveraswellasitsresponsetoanthropogenicdisturbancesandenvironmentalchanges(Hanetal.2019).However,thepredictionofsoilEAiscomplicatedsinceenzymesarenotevenlydistributedintheenvironment(Baldrian2014).RelativelyhighspatialvariabilityofsoilEAhasbeenobservedevenwithinasinglefeldbecausesoilEAareregulatedbymultiplebioticfactors(e.g.,vegetationandmicrobialcommunitycomposition)andabioticfactors(e.g.,soilphysico-chemicalpropertiesandmicroclimate).Inrecentdecades,anumberofstudieshavebeenconductedtoexplorethespatialvariationsofsoilEAandtheirdeterminantsataglobalorregionalscale.Basedonthesestudies,soilpHandSOMcontentwereidentifedasthemostimportantabioticfactorsafectingsoilEA,especiallyforsoilhydrolaseactivity(Sinsabaughetal.2008;KivlinandTreseder2014;Xuetal.2020).However,thespatialvariabilityofsoilEAwithinecosystemshasbeenlargelyneglected(Banerjeeetal.2016;Stursovaetal.2016).Whetherobse,ationsatlargescalescouldapplytoasingleecosystemisstillanopenquestion.AlthoughafewstudieshaveinvestigatedthespatialheterogeneityofsoilEAwithinecosystemsandthedeterminants,theseconclusionsareindebate.Forexample,Boeddinghausetal.(2015)reportedthatsoilpHwasanimportantdeterminantofspatialdistributionofEAinagrasslandecosystem,whilestudiesconductedinforestecosystemsandinagrassland-woodlandecotonefoundthatsoilpHwasnotspatiallyconnectedwithsoilEAbecauseoftheverylowvariationsofpHobservedinthesestudies(Banerjeeetal.2016;tursovaetal.2016).Mayoretal.(2016)investigatedthespatialdiferencesinsoilEAlevelsbetweenvegetationpatchesandinter-patchesinashrublandecosystem,andfoundthatvegetationcanopycouldafectsoilEApatternsthroughrhizosphereefectsandsubstrateinputbyIitterfall(Dornbush2007;Brzosteketal.2013;Fengetal.2019).Bycontrast,vegetationpropertiesdidnotSignifcantlyinfuencesoilEAinaforestecosystem(Slursovdetal.2016).Meanwhile,soilmicrobialbiomass(MB)andcommunitycompositionhavebeensuggestedasdirectregulatorsofsoilEAspatialpatternssincetheproductionofaspecifcenzymeisinducedbysomeparticularspecies(BaldrianandSnajdr2011;Bowlesetal.2014),butexceptionswerealsofoundinotherstudiespartlydueIothatalargeportionofsoilMBwasmetabolicallyinactive(Bocddinghausetal.2015;Stursovdetal.2016).Inaddition,soilpHwasfoundtobecorrelatedwithCellobiohydrolaseandchitinaseratherthan-and-glucosidasesinagrasslandecosystem.AllthesecontradictoryresultsindicatedthattheefectivenessofbioticandabioticvariablesinregulatingsoilEAwithinecosystemswouldvarydependingontheecosystemtypeorenzymetype.Despiteofthesestudies,onlyfewstudiescomparedtheefectsofsoilphysico-chemicalvariables,vegetationproperties,andmicrobialcommunitytogetheronthevariationsofsoilEAwithindiferentecosystems.Moreover,asmultiplefactorsareinterrelated(Walleniusetal.2011),therelativecontributionsofthesefactorstosoilEAspatialpatternshavenotbeenwellinvestigated.SoilEAcanbeexpressedindiferentformsincludingtheabsoluteactivity(i.e.,activityperunitofoven-drysoilmass)andthespecifcenzymeactivity(i.e.,activityperunitofSOMorMB).ThespecifcEAcanbeusedtoeliminatetheimpactofSOMorMB,andtestwhethervariationsofsoilEAcanoccurindependentlyofvariationsofSOMorMB(RaiesiandBeheshti2014).Todate,muchlessisknownabouthowthespecifcEArespondspatiallytootherabioticandbioticfactorsatsmallscaleswithindiferentecosystems.Inaddition,therelativeabundanceofsoilC-andN-hydrolyzingenzymes(i.e.,enzymaticC/Nratio),namely,enzymestoichiometry,exhibitsthepotentialtorefectthebiogeochemicalequilibriumbetweenmicrobialCandNdemandsandnutrientavailabilityoftheenvironment(Sin-sabaughetal.2009;Mooshammeretal.2014).Thus,spatialvariationsofenzymaticC/NratiowithinecosystemscouldprovideafunctionalassessmentoftherelativeresourcelimitationsofmicrobialmetabolismandtherelativeratesofSOMdecomposition(Sinsabaughetal.2009;Liaoetal.2021).Previousstudiesconcerningenzymestoichiometrypatternshavebeenmainlyconductedatalargeorglobalscale(Sinsabaughetal.2008;PengandWang2016).ThesestudieshaveshownthatratiosofC-andN-hydrolyzingenzymeactivitiesconvergedon1:1.Itislessclearifthepatternofenzymestoichiometryataglobalscalecanbeappliedwithinecosystems.Inthepresentstudy,weinvestigatedspatialvariationsofsoilC-andN-hydrolyzingenzymes(defnedasenzymesthatcontributetothehydrolysisoforganicCandNcompoundsinsoils)withintwotypicalaforestedlands(woodlandandshrubland),aswellasthefactorsthatbestregulatingthesevariations.WehypothesizedthatthespatialvariationsofEAwithinecosystemscouldbeexplainedbythecombinationoffactorsrelatedtosoilenvironment,vegetation,andmicrobialcommunity,butthemajordeterminantsofthesevariationswouldVarydependingontheecosystemtypeandenzymetype.Inaddition,toexploretheallocationofC-andN-hydrolyzingenzymes,wealsohypothesizedthattheenzymaticC/Nratiowithinecosystemsshouldbesimilartothatataglobalscale,whichconvergedon1:1.MaterialsandmethodsStudysiteandsamplingdesignThestudywasconductedintheexperimentalareaoftheWulongchiResearchStation(32o45,N,111o13,E),HubeiProvince,China.Primaryforestsinthisregionwereconvertedtocroplandsabout70yearsagoasaresultoftheresettlementofinhabitants(LiandZhang2008).Largeareasofcroplandswerelaterconvertedtoopenareaswithnovegetationcoverduetointensivelanddegradation.Sincethe1980s,aforestationhavebeenimplementedinthisregion(Zhuetal.2010).Asaresult,mostoftheopenareashavebeenconvertedtowoodlandandshrublandplantations.Thetwoaforestedlandswereunderdifferentnutrientregimes,namely,thatthewoodlandsitewasplantedwithconiferoustrees(Platycladusorientalis(Linn.)Franco,characterizedbyhighC/Nratio),whiletheshrublandsitewasplantedwithleguminousN-fxingshrubs(Sophoradavidii(Franch.)Skeels).Thisdiferenceinlitterqualitybetweenthetwosystemsresultedindifer-entnutrientregimesanddiferencesinsoilenvironmentandmicrobialcommunity(Table1).Managementsuchasfertilizationandirrigationontheseareashasbeenminimal.BothoftheaforestedsystemsthatdominatedbythetwospeciesalsowidelydistributeinnorthernChina.AstandofIO×10mconsistingof2×2mgridcellswaslaidoutineachofthesitesincludingwoodland,shrubland,andtheopenarea(i.e.,control)inApril2017.Surface(0-10cm)soilsamplewascollectedusingacoreaugerateachnodeofthesegrids,resultingin36samplesperstand(Fig.1).Beforesoilsampling,wecompletelycollectedtheabovegroundlitterusinga0.2×0.2mframeateachnode.Alllivingrootsineachsoilsampleswerecarefullyseparatedfromdeadrootsandwashed.Litlerandlivingrootswereovendriedat65toaconstantweighttoobtainthelitterandlivingrootbiomass(gm2).Allplantsthatlocatedinsideandaroundthe100-m2standwithdiameteratbreastheight(DBH)1cmweremeasured,andtheirgeographiccoordinateswererecorded(Fig.1).SoilanalysisEachfreshsoilsamplewassievedwitha2-mmmesh.Aportionofeachsoilsamplewasfreezedriedforthemeasurementofphospholipidfattyacids(PLFAs),andanotherportionofsoilsampleswerestoredat4forthedeterminationofsoilEAwithin72h.Theremainingsoilswereairdriedforthedeterminationofothersoilproperties.Soilmoisturecontent(SMC)wasobtainedgravimetricallybyOven-drying2()goffreshsoilat105toconstantweight.SoilpHwasmeasuredaftershakingasoil-watersuspension(1:2.5)for30minwithadigitalpHmeter.Soilorganiccarbon(SOC)andtotalnitrogen(STN)concentrationsweredeterminedonanelementalanalyzer(ThermoScientifcFlash2000HT,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 (%)19.3a2110.8b354.3c22SoilON19.4a2811.5b139.4c16SOC (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 PLFA50.57a557.27b595.48c73ACT 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, 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, actinobacteriaSoilrecalcitrantC(RC)andN(RN)concentrationswereobtainedbyacidhydrolysis(seemoredetailsinXuetal.2015).Briefy,500mgofair-driedsamplewastreatedwith25mlof2.5MH2SO4.Theresiduewasrecoveredbyrepeatedcentrifugation,andthentreatedwith2mlof13MH2SO4overnight.Theremainingresiduewasrecoveredagainasdescribedaboveandthendriedat60Eastin(mlFig.1SoilsamplingschemeandtreedistributioninthethreekindsofecosystemsintheDanjiangkouReservoirarea.Blueasterisksrepresentsoilsamplingsites.RedcirclesintheWoodlandrepresentPlat-ycladusorientalis(Linn.)Franco.RedandgraycirclesintheshrublandrepresentSophoradavidii(Franch.)SkeelsandViiexnegundoL.var.Cannabifolia(SiebetZucc.)Hand.-Mazz.respectively.Thesizeofthecircleinthewoodlandandshrublandisproportionaltotreediameteratbreastheightundshrubbasaldiameter,respectivelyformeasuringRCandRNontheelementalanalyzer.TherecalcitranceindicesforCandN(RICandRIN,respectively)werecalculatedastheratiosofRCandRNtototalCandN.ThemicrobialbiomassandcommunitystructurewereassessedbyusingPLFAanalysis(BossioandScow1998)(seemoredetailsinWuetal.2017).SpecifcPLFAmarkerswereusedtoquantifydiversetaxonomicgroups,includinggram-positivebacteria(G)gram-negativebacteria(G-),actinomycetes(ACT),arbuscularmycorrhizalfungi(AMF),totalfungi(F),andtotalbacteria(B)accordingtoXuetal.(2015)andareshowninTableSl.Amodifedmethodbasedonfuorescent-linkedsubstratemicro-plateprotocolwasusedtomeasuretheactivitiesofenzymes(including-glucosidase(BG),/V-acetyl-glucosaminidase(NAG),andleucineaminopeptidase(LAP)insitupHconditionsandtemperature(Smithelal.2016).Inthepresentstudy,weselectedthesesoilC-andN-hydrolyzingenzymesduetothefollowingreasons:(1)hydrolysishasbeenconsideredmoreimportantthanoxidationinSOMmineralizationprocesses(Nannipierietal.2012),(2)theiractivitiesareusuallymuchhigherthanotherhydrolasesacrossdiferentecosystems(Bowlesetal.2014;Schimeletal.,2017;Zhangetal.2019),and(3)theratioofthethreeenzymes(InBG:ln(NAG+LAP)wasgenerallyusedtorepresentenzymestoichiometry(Sinsabaughetal.2008;PengandWang2016).Inbrief,1gofsoilwashomogeneouslydispersedin90mlofsodiumacetatebufer.ThepHofthebuferwasadjustedto8.0.Thepreparedsoilsuspension,standardsolution,andfuorescentsubstratesolution(200Mintotal)weresuccessivelyaddedintoa96-wellmicroplate(WhatmanInc.,FlorhamPark,NJ).Thecompounds7-amino-4-methyIcoumarin(MUC)and4-methylumbclliferone(MUB)wereusedasthestandardreferencesforLAPandIheremainingenzymes,respectively.Themicroplatewasincubatedat25for3.0h,andthen50lof1MNaOHwasaddedtostopthereaction.Thereleasedfuorescencewasdeterminedusingamultifunctionalmicroplatereader(TecanInfniteM200pro,Salzburg,Austria).Thewavelengthofexcitationusedwas360nmandtheemissionusedwas450nm.UnitsforabsoluteEAwereexpressedasnanomolesoffuorescenepergramofdryfractionperhour.EAwerenormalizedbyMBandSOMtoobtaindifferentspecifcEA(EA:MBandEA:SOM).CalculationandstatisticsWeusedaproximityindex(treeinfuencepotential,IP)toquantifytheinfuenceoftreedistributiononsoilEA(SaetreandB熊th2000).ThisindexrefectsthecombinedinfuenceofneighboringtreesonsoilEAateachsamplingpoint,andisexpressedusingEq.(1):P=iDBHie×p(-cdi)(1)whereDBHiisthediameteratbreastheightorbasaldiameterofplantiinmeters,CiSascalingcoefficientandwassett