LUIS CARLOS COLOCHO HURTARTE
PLANT NITROGEN STATUS DRIVING SOIL ORGANIC
MATTER MINERALIZATION IN THE RHIZOSPHERE
Dissertação apresentada à Universidade Federal de Viçosa, como parte das exigências do Programa de Pós-Graduação em Solos e Nutrição de Plantas, para obtenção do título de Magister Scientiae.
VIÇOSA
MINAS GERAIS
– BRASIL
ii AGRADECIMENTOS
À Universidade Federal de Viçosa e ao Departamento de Solos, por possibilitar a realização deste curso de mestrado.
À CAPES, FAPEMIG, CNPq, SIF e ao Grupo NUTREE pelo auxílio financeiro
Aos Professores Ivo Ribeiro da Silva e Leonardus Vergutz, pelos ensinamentos, discussões e confiança acompanhados de momentos de descontração nestes anos.
Ao Prof. Mauricio Fontes e a Rogerio Faria pelo incentivo e ajuda no início da graduação. Aos demais Professores do Departamento de Solos, por contribuírem para minha formação, em especial Victor Hugo, Jaime Mello, Júlio César Lima Neves, Nairam Félix de Barros e Maurício Fontes.
Aos funcionários do DPS e da Pós-graduação.
Aos técnicos do Laboratório de Isótopos Estáveis (LIE) João Milagres e Humberto, pela constante ajuda, paciência e pela importante contribuição para a realização deste trabalho. Aos estagiários pelas contribuições na execução dos experimentos, em especial a Gabi e Crock (Rodrigo).
Aos colegas da Pós-graduação e do LIE, em especial Ricardo, Fernanda Caparelli, Daniela, Marina, Ana, Helen, Gabriel (Tcha), Medina, Ivan, Luis Bola, Gustavo, Chico Bento, Matheus Barreto, Rafael, Crock e Luiz.
Aos membros da banca Rogerio, Samuel e Leonardus pelas sugestões que levaram à melhoria da qualidade do trabalho.
Aos amigos do intercambio em especial Bruno, Daniel, Marco, Lobão, Fábio, Maria, Thera e Alexandre.
A meus queridos pais, Jose Luis Colocho e Maria Margarita e minha irmá Ana Elena pelo constante apoio e incentivo, são vocês que fazem tudo valer a pena.
Aos meus amigos/irmãos de Viçosa Rodrigo, Álefe, Conrado e Josimar, agradeço pela força nos momentos difíceis e pelas risadas no dia a dia.
iii BIOGRAFIA
LUIS CARLOS COLOCHO HURTARTE, filho de Jose Luís Colocho Ortega e Maria Margarita Hurtarte, nasceu no dia 06 de dezembro de 1989, em Viçosa/Guatemala – MG.
Em dezembro de 2007 concluiu o ensino médio no English American School em Guatemala e em agosto de 2009 foi aprovado em primeiro lugar por transferência para a Universidade Federal de Viçosa no curso de Agronomia, concluindo em janeiro de 2015.
Entre agosto de 2012 e marco de 2014 foi bolsista do programa Ciência sem
Fronteiras/CNPq na modalidade “graduação sanduiche”, na Universidade Técnica de
Munique (Technische Universitaet Muenchen), sob orientação dos Professores Thilo Rennert e Joerg Prietzel.
Em marco de 2015, ingressou no Programa de Pós-graduação em Solos e Nutrição de Plantas da Universidade Federal de Viçosa, em nível de Mestrado sob orientação do Professor Ivo Ribero da Silva.
A mediados de 2016 foi aprovado nos programas de doutorado completo tanto Instituto Max Plack de Biogeoquimica pela International Max Planck Reseach School on Global Biogeochemical Cycles e na Universidade Técnica de Munique.
Em janeiro de 2017 submete-se a a defesa de com a dissertação titulada: “Plant Nitrogen status driving soil organic matter mineralization in the rhizosphere”.
iv SUMÁRIO
RESUMO ... vi
ABSTRACT ... viii
INTRODUCTION ... 1
MATERIALS AND METHODS ... 3
Experimental set-up ... 3
Bulk analyses ... 6
Rhizospheric soil solution analysis ... 7
Root image analysis and metabolic profiling ... 7
MAOM molecular characterization using TMAH-GC/MS ... 8
Synchrotron XRD analysis coupled to EGA-MS ... 9
Statistical analysis ... 10
RESULTS ... 10
Shoot and root changes due to N-deficiency ... 10
Changes in soil properties ... 12
Synchrotron-based XRD and EGA-MS ... 13
Molecular characterization of organic compounds in the MAOM fraction... 14
DISCUSSION ... 15
Effects of N-fertilization on root growth and physiology ... 15
Rhizospheric priming as function of plant N status ... 17
Implications for C dynamics ... 21
FIGURES AND TABLES ... 23
Table 1 ... 23
Table 2 ... 23
v Fi gure 2 ... 24 Fi gure 3 ... 25 Table 3 ... 26 Table 4 ... 27 Fi gure 4 ... 28 Fi gure 5 ... 29 REFERENCES ... 30 SUPPLEMENTARY MATERIAL ... 37
Suppl em ent ar y Fi gure 1 ... 37
Suppl em ent ar y Table 1 ... 38
Suppl em ent ar y Fi gure 2 ... 38
Suppl em ent ar y Table 2 ... 39
Suppl em ent ar y Fi gure 3 ... 40
Suppl em ent ar y Fi gure 4 . ... 41
Suppl em ent ar y Fi gure 5 ... 42
Suppl em ent ar y Fi gure 6 ... 43
Suppl em ent ar y Table 3 ... 44
Suppl em ent ar y Table 4 ... 44
Suppl em ent ar y Table 5 ... 47
vi RESUMO
HUR TAR TE, Luis C arl os Col ocho , M.S c., Universidade Federal de Vi ços a, janei ro de 2016 . Plant Nitrogen status driving soil organic matter mineralization in the rhizosphere. Orientador: Ivo Ribeiro da Silva. Coorientadores: Leonardus Vergutz e Mauricio Dutra Costa
Os fat ores que regul am a dinâmi ca do Carbono (C) e Nit rogênio (N) do s olo na rizosfera s ão ai nda pouco compreendidos. A mineraliz ação de C na rizos fera pode s er fortement e i nfl uenciada pelo estado nutri ci onal da pl anta, a concent ração de CO2 na atmo sfera e a tem peratura do ambiente, ent re outros. Em est e estudo, avaliamos o st at us nut ri cional de N em pl ant as de Eucal yptus s pp. e s ua influênci a na di nâmi ca do C e do N n a rizos fera. Realizam os um experim ento us ando um r hizobox dividido em dois com partim ent os. No compartim ent o de cim a pl ant as foram culti vadas em arei a l avada e supridas com um a s olução nut ritiva c ont endo todos os nutrientes e a mes m a sol ução porem s em N . No com partim ent o i nferior o contat o das raíz es com o sol o foi limit ado usando um a mem brana de n yl on com abert ura de 5 µ m. Obs ervamos uma maior razão rai z:parte aérea e maiores concent rações de C O2 no sol o das pl antas com defi ciênci a de N. As raíz es das plant as deficientes em N, apres ent aram m aiores concent rações em rel ação as plant as não d efi ci ent es em N, de ci trato e t all os e, e m enores concent rações de sucros e e ami noácidos . A anális e de C e N da fração de mat éri a orgânica li gada aos mi nerais , junto com os dados obtidos pel a termoquimoli se indi cam um aumento na m ineraliz ação de C e um a modi ficação na dinâmica do N. Devi do a impossi bilidade de contat o físi co direto com o s olo, pel a pres ença da membrana de n yl on, a úni ca form a de modi ficar o s olo s eria então pel a exsudação de compostos pel as raízes . O contrast ant e conteúdo de am inoácidos e aç úcares na raiz, junt o com os dados do extrat o da solução do sol o e de m ineralização de C, indi ca que a com posi ção dest es exsudatos di feri u em razão da defi ci ênci a de N. Enquant o as pl ant as defici entes em N exsudaram m ais áci dos orgâni cos, as plantas com ótimo st atus nut ricional foram capazes de exsudar com post os energeticam ent e ri cos. Os dados de δ1 3C da matéri a orgâni ca li gada aos
vii minerais indica que as pl ant as deficientes em N afet aram um m aior volume de solo que as pl antas supridas de N. Tudo ist o mos tra que, di ferent es mecanismos de efei to priming foram dominant es, dependendo do s tat us nutrici onal da pl ant a. Em pl ant as defi ci ente de N, a minerali zação de C no
solo foi dominada pelo mecanismo chamado de “mineração de N”, enquanto
no sol o das pl ant as supridas de N o mecanism o dominante foi a
“estequiometria microbiana”. Este trabalho demostra pela primeira vez, ao
noss o s aber, a atuação de diferentes m ecanismos de efeit o pri ming n a m esm a planta, s obre di ferente st atus de N . As sim res sal tando, a i mportânci a do manejo de nut rient es na di nâmi ca do C da rizos fera.
viii ABSTRACT
HUR TAR TE, Luis C arl os Col ocho , M.S c., Universidade Federal de Vi ços a, J anuar y, 2016. Plant Nitrogen status driving soil organic matter mineralization in the rhizosphere. Adviser: Ivo Ribeiro da Silva. Co-advisers: Leonardus Vergutz and Mauricio Dutra Costa
The factors that regulat e the d ynamics of soil Carbon (C) and Nitrogen (N) in the rhizosphere are still poorl y unders tood. The soi l C mineraliz ation in the rhiz osphere can be heavily influenced by plant’s nutritional status, atmospheric C O2 concent rati on and tem perat ure, am ong others. In thi s stud y, we ass ess the influence of Eucal ypt us s pp. N st atus on the C and N d ynami cs in the rhizos phere. We perform ed an experim ent us i ng t wo compartment rhizobox. In t he upper compartment, pl ants were culti vated i n washed s and and suppli ed wit h a soluti on containing all nut rients or all nutri ent s but N. The lower compartm ent li mited the cont act of the root s wit h t he s oil us ing a 5 µ m m esh n yl on m embrane. W e obs erved a hi gher root -shoot ratio for t he N defi cient plants and an increas e i n it s s oil CO2 concentrati on. The roots of
the –N planted treatment had higher concentrations of citrate and tallose and
lower concent rat ion of sucros e and am inoacids , when compared to the +N planted t reatm ent. The C and N anal ys is of the mineral associ ated organi c matt er fracti on, t oget her wit h the therm ochem ol ysi s data s howed an increas e in C mineralizati on i n bot h pl ant ed treat ment s and changes i n N d ynam i cs . As the roots had no ph ysi cal cont act with t he s oil due to the n yl on m embrane, the changes i n the s oil must have been cons equence of root exudation. The contrasti ng s ugar and aminoacid root content, t ogether with the cit rat e concent ration i n soil sol ut ion extract and the C mineraliz ation dat a, indi cat e that exudate composition changed due to the pl ants N st atus. The dat a indi cat es that the pl ants in the –N treatment exudated more organic acids than the pl ants of t he +N t reat ment. S t ill the exudat e com p ositi on of the plants wit h the +N t reatm ent m a y had a hi gher energeti c content and t hus affected di fferentl y t he s oil mi crobi al communiti es. The δ1 3C dat a indi cat e that the N defici ent plant s affected a hi gher volum e of s oil than the pl ant s of the +N t reat ment. All thi s together shows different primi ng m echanis ms were
ix dominant due to the pl ants N st atus. As the plants were N defi cient, the mineralization of soil C was driven by the “N-mining” mechanism while in
the soil of the +N planted treatment the dominant mechanism was “microbial stoichiometry”. This work demonstrates, to our knowledge, by the first time
using the s am e plants, di fferent priming mechani sms due to the pl ant s N stat us. Thus hi ghli ghting, the i mport ance of plants nut ri ent managem ent in the rhizosphere C d ynami cs .
1
Th e glob al t errest ri al carbon (C) b al an ce i s m ain l y dri v en b y fores ts (J obb ág y an d J ack s on, 2 000 ), whi ch to get her cov er ~30 % of th e bo real, temp erate an d tropi cal l and su rface (Bon an, 200 8). The C balan ce can b e affected b y t he eco s yst em’s h ealth (Trumbo re et al., 2 015 ) (eg. nut ri ent defi cienc y, C O2 ferti lizatio n, di seas es , et c), whi ch in tu rn is affect ed also b y clim at e ch an ge. Thi s o ccu rs b ecaus e cli mat e in flu ences s ev eral im po rtant vari abl es for t he eco s yst ems h ealth s uch as t emp erat ure, C O2 and N b al an ce. As it has recentl y b een expos ed (He et al., 2 016 ) soils mi ght n ot b e an effectiv e C s ink as it was previ ousl y t ho u ght. Ho wev er s oils stil l ho ld a m ajo r rol e as a s ou rce o f variou s green hou se gas es (Am undson et al., 2 015 ). Therefore, i t i s imp ort ant t o as ses s and correctl y mo del t h e resp ons es o f fo rests to cli mat e chan ge and h ow t his feedback will affect the t errest ri al carb on b al an ce (Bon an, 2 008; Heim ann and R ei chs tei n, 20 08; Trumb ore et al., 2 015 ).
Fo rests co ntribut e to the n et C bal an ce b y eith er acti n g as a so urce or a sink of C O2. Wh en actin g as a si nk, a large part of th e fores t C can be sto red in th e soi l. The mai n inp ut o f C to th e soil are the ro ots to geth er wit h microbi al d eriv ed co mpoun ds (J ones et al., 20 09; Kall enb ach et al ., 2 016; Rass e et al., 200 5) esp eci all y i n th e s ubsoil (Rum pel and Kö gel 9Knabn er, 2011 ).
The t rans fer C t o th e soil can in creas e or in hibit s oil o rgan ic m atter (SOM ) min eral izati o n. This p henom en a is k no wn as rhizos p here p rimi n g
2
(Kuz yakov , 20 02). The root s can p rom ot e th e p rimin g effect b y ex ud atin g different organic co mpoun ds (Pat erso n et al., 2 007 ). Th e roots ch an ge it s ex udate comp ositi on as a ph ys iol o gi cal resp ons e o f t he pl an ts to abioti c and bioti c st ress es, fo r inst an ce nut ri ent l imitati ons (Dijk stra et al., 20 13; Kuz yako v, 200 2; Wi ed er et al., 20 15 ).
In a ch an gin g clim at e pano ram a, th e n utri ent req uirem ent s o f fo rest m a y shift an d wh en defi cient , pl ants m a y s caven ge fo r nut ri ent s b y mi neralizin g the S OM (Hei mann and R eichst ein , 2 008 ). This is especi all y tru e fo r N, whi ch is a limi tin g nut ri ent in several ecos ys tem s. An i ncreasin g con cern on th e N9 clim at e feedb acks h as d rawn i nterest in coupli n g the effects o f N d efi ci en c y to clim at e vari abl es , su ch as CO2 i ncrease (Hun gate, 200 3). Still up to this da y, few experim en ts are abl e t o co nfi den tl y, s ep arat e th e effects o f N defi cienc y in th e pl ants ex ud at es, an d its effect on SOM min eralization . Onl y a h and full of experi ment s most o f th em u sin g art ifi ci al ex udat es h av e t ackl ed this probl em (Drak e et al., 2 013; M ei er et al ., 2017).
In th e t ropics th e Eucal yp t forest s are wit h in on e o f th e mo st p rodu ctiv e ecos ys tems in t he gl obe (St ap e et al ., 2 0 04). Alt hou gh mu ch is kno wn ab out the tree, a con und rum h as emerged regardin g t h e l ack of resp ons e to N fertiliz atio n an d it s hi gh N requi rements. Th e cu rrent m ost accept ed h ypot h esis is th at th e trees promot e SOM to access mo re N. Th is ma y be true as Eu cal yp tus ’ ro ots ex udate l arge amou n ts o f organi c acids, whi ch in creas e und er abioti c st resses su ch as hi gh Al co ncent rations (Silv a et al ., 200 4).
3
Here we att empt t o t ak e a st ep furth er i n the N cl imate feedb acks b y unrav eli n g th e rhizo sph eri c N9d riv en p ri min g processes, th ro u gh a rhizobox experim ent . Th e main obj ectiv e o f t his stu d y was t o as ses s th e p ossi ble mech anism s for rhiz osph eric primin g effect in a repres ent ati ve t rop ical s oil
culti vated with . We aim ed to achi ev e th is b y, ass es sin g (i )
the effects of N fert ilizat ion in t he root ’s trait s and p h ysio lo g y. (ii ) Revi ewin g th e ch an ges i n soi ls p roperti es such as mi neralo g y, pH, C and N con tent and o rgani c acids in i ts s oil solut ion, and fin all y b y (i ii) relatin g th e plants N9st atus to chan ges i n t he mo lecul ar com posit ion o f S OM in th e rhizos ph ere.
The experim ent was co ndu ct ed usi n g a rhizobox s ys t em d esi gned b y Wenzel et al ., (2 001 ). This s yst em is co mpos ed of two ph ys icall y separat e com partm ent s: an upper comp artm ent cont ainin g wash ed < 2 mm sand in whi ch th e pl ant s were cult iv ated and a l ower com partm ent cont ainin g soil . The up p er comp artm ent h as a narrow slit in it s b ottom , th ro u gh whi ch pl ant root s can v erticall y pen et rate i n t he l ower com partm ent . Th e cont act of t he root s with th e soil in th e l ower comp artm ent is limit ed b y a n ylon m em bran e with a p ore s ize o f 5 µm (Tegap e). See s ch em ati c rep res entati on of t he rhizob ox in t he Sup p lem ent al Fi gu re 1.
4
The rhizobox s ys t em mad e possi bl e t o m o dif y t he n utri tion al s tatu s of the pl ants wit ho ut m odif yi n g th e nut ri ent co nt ent of th e s oil. This was don e b y ap pl yi n g to th e up per co mp artm ent differen t nut rient sol utio ns, cond uct ed b y gl as s fi ber wi cks (Fib erseals ). We perfo rmed a facto ri al experim ent with two facto rs with two level s each .
The fi rst fact or was the p lant effect , thu s rhizob ox es were ass embl ed with pl ant s (+Pl) o r witho ut pl ants (9Pl ). The p lants us ed fo r t he exp eriment were comm ercial l y avail abl e 3 mont hs ol d
cl on al s eedlin gs. Th e s econd facto r was th e N effect, a com pl ete modi fied Cl ark solu t ion (+N, with a label ed 1 5N 9 2 % APE) or an in comp let e one (9N) was suppl ied t o th e upp er comp artm en t, d etails of t he Cl ark soluti ons are in t h e Supp lem entar y T abl e 1. Th ree rep et itions o f each treatm ent were ass embled, a s ch eme of the experim ent can be fou nd in t he Suppl em ent ar y Fi gu re 2 .
The lo wer comp artm ent was fil led with so il coll ected from a cu ltivated
trop ical p ast ure ( 20 years ) in Paula C and ido , Min as
Gerais – Brazil (2 0° 5 2’ S, 42 ° 5 8’E). This p astu re fi eld (
is a C 9C 4 sp ecies and it s cul tivat ion after a C 9C3 nati ve fo rest lead t o an in creas e i n th e s oil 1 3Cδ from 927 t o 916 .75 ‰, th us allo win g u s t o track t he root d eriv ed C i n th e s oil. Th e soil was cl assi fi ed as a Ferras ol (T yp li c Haplu dox acco rdin g t o t he U.S. S oil Tax on om y) l oam y tex tu re, pHH2O of 5.2 4, t ot al o rgan ic carbon o f 2 5.4 g/ k g of C (± 0 ,92 ).
5
A soil 9air prob e was placed in t he lower comp artm ent, whi ch consist ed of a sil icon e rubb er tub e. Th e tub e was 20 cm lo n g, with a 5 mm int ern al diam et er an d a 1 m m width . It was accommod at ed s pirall y in th e cent er o f the box . The sili con tub e was co nn ected t o a 15 cm copp er tub e, t hat cro ss ed thro u gh and proj ect ed out o f th e upp er b ox . Th e copp er tub e’s en din g was kep t s hut u sin g a th ree wa y s topcock (Suppl ement ar y Fi gu re 2 9 Kamm ann et al., 2 001 ).
The experim ent too k place i n a gro wth ch amber (t emperat ure of ̴2 5 °C and air rel ati ve hu midit y o f ̴ 6 0%). Plant s were gro wn in t h e upp er com partm ent , and after 2 0 d a ys th e com plet e rh izobox was assem bl ed. Th e rhizob ox es were k ep t each ins id e a 30 L pol yetil en e b lack b ox in order to avo id l i ght dis tu rb an ce to th e ro ots. Pl ant s were cultiv at ed for 30 d a ys aft er the ro ots st art ed to p en et rat e in t he lo wer co mp artm ent.
The da y b efore u nm ounti n g th e experim ent , rh izobox es were cov ered usin g an acr yli c ch amber and puls e9l ab el ed with 1 3C (M achad o et al ., 2 011 ). The follo win g da y, t he SP AD in dex o f th e p lant mi ddl e l eav es was m easu red usin g a Chl oroph yl l met er SPAD9502Pl us (Koni ca Mi nolt a – J ap an ).
The s oil ai r s ampl es were t ak en usi n g a 5 ml s yri n ge, tran sferred to previo usl y ev acu ated 12 ml ex et ei ners . Th e sam pl es were anal yzed fo r C 9C O2 and 13C/12C of C9CO2 relative to the PDB internal standard in an
(ANCA9GSL, 20920, Sercon, Crewe, UK). Then, the plants were tak en out o f th e p ol yet h yl en e box es and the fo rm ed root m at in th e lo wer
6
com partm ent was p h oto graph ed , coll ect ed and imm ediat el y freez ed in N2 an d kep t in an ul trafreezer at 980 °C until further an al ys is.
The ab ov e groun d mat eri al was als o collect ed , separat ed i n gro wn befo re (old ) and aft er (new) th e experim ent st art ed. Th e s ampl es were d ri ed in a fo rced ai r furn ace, wei ghted, mill ed an d k ept i n ai rti g ht box es until fu rth er an al ysi s.
Fin all y, th e rhizob ox es were op en ed and t he so il moist ure was p rob ed usin g a s enso r (EC 95, Decago n, Pu llm an, WA). Th e s oil was s ectio n ed i n three di stan ces p aral lel t o t he root mat (0 9 3 mm, 39 6 m m and 6 91 5 mm ) usin g a m odi fi ed mi crotom e from Fitz et al., (200 3). Th e samp les were divid ed in t wo, on e s ubs ampl e was us ed t o ex tract th e soil sol ution acco rdin g to Pérez et al. (20 02 ). The rem aini n g so i l sam ple an d th e so il soluti on were kep t in an ul trafreezer unt il fu rth er an al ysis.
The s oil s ampl es were freez e9dri ed an d di vid ed in two, th es e were us ed to (i ) meas ure th e p H in wat er accordin g t o (P ans u and Gau the yro u, 2 006 ) and (ii) to p h ysi call y fraction at e th e S OM according t o C amb ard ell a and Elliott (19 92 ). The fracti ons were d esi gn at ed as p arti cu lat e o rgani c m atter (POM >5 3 µm) an d mineral asso ci at ed o rgan ic m att er (M AOM < 53 µm ). Th e fractio nat ion was p erfo rm ed b y di sp ers in g 3 g of the sam ple in so dium hex am et aph osp hate, and si ev ed at 53 µm. Th e two fracti ons were d ried in a fo rced air ov en at 50 °C, an d k ept in airti ght b ox es until furth er anal ysi s.
7
The OC, N cont ent and 1 3C/1 2C and 1 5N/1 4N ratios o f t he MAOM fractio n was d et erm i ned. Th e chan ges in s hort 9ran ge9ord er and organi c b oun d Fe and Al ox yh yd rox ides were ass es sed b y i nd ep en dent ox alate an d p yroph osp hat e ex tractio n and anal yz ed b y atomi c ab so rptio n sp ectrom etr y (Sh an g and Zelazn y, 2008 ).
The ex tract ed s oil s o lution was freeze dri ed an d recon stitut ed in 1 ml of ult ra9pu re wat er and pass ed th rou gh a 0.45 µm m emb rane. An al iqu ot of 0.5 m l was dil ut ed i n 2 ml o f ult rap ure water and anal yz ed fo r organi c acid s b y io n ex ch an ge chromat o graph y (Si lv a et al., 2 004 ).
The p hot o graph ed roots were an al yz ed usin g th e Gi aR oots soft ware (Galk ovs k yi et al ., 2 012 ), th e croppin g to ol was used to s elect th e root m at area and th e s cal e was m anu all y d efin ed . Th e root identificati on param et ers were ch os en for each p hot o i ndi vidu all y usi n g th e do ubl e adap tiv e image thresh oldi n g. Th e co mput ed trait s were: average root widt h, ro ot area, l en gth, surface area and s p ecific root len gth .
The ex tractio n and i den tifi catio n o f com poun ds p rodu ced b y the ro ots in th e l ower box , wit h po ssibl e i nvo lv ement in th e rhizos ph eri c prim in g effect was m ad e foll owi n g t he m et hod from ( Lis ec et al., 20 06 ). Bri efl y, root tiss ue (10 0 m g) were hom o gen ized with liq uid nitro gen and ex tract ed i n 1.4 m L of meth an ol, an d 60 µL of int ern al st an dard (0.2 m g rib itol/m L wat er) was
8
subs eq uentl y add ed as a qu anti fi cation st and ard. The mix ture was ex tract ed fo r 1 5 min at 70 °C and mix ed vi gorousl y wit h 1 .4 m L o f wat er. In order t o sep arate pol ar and n onpol ar m et abol ites, 750 µ L o f chlo ro fo rm was add ed to the mix tures . Aft er cent ri fu gati on at 2,2 0 0 , th e upp er m eth anol/wat er p hase was t ak en ad red u ced to d r yn es s i n vacu u m. Th e s am pl e was resu sp end ed and deriv atiz ed in 40 µ L of 2 0 m g/m L metho x yamin e h yd rochlo ri de in p yridi ne at 37 °C fo r 1 20 min . Afterwards , th e ex t ract was treat ed wi th 60 µ L o f N9 meth yl 9N9[thri met h ylsil yl]trifl uo ro acet amide at 37 °C for 30 min. A st an dard alk an e mix ture (n 9d odecan e, n 9p ent ad ecan e, n 9no nadecan e, n9d ocos an e, n 9 oct acos e, n9dot raco ntane an d n 9h ex at ri acont an e) dissolv ed in anh yd rous p yri din e (0.0 29 % [v/ v]) was ad d ed b efore trim eth yl s yl ati on for ret ention tim e con tro l. S amp le vol u mes of 1 µ L were an al yz ed i n an S him ad zu QP 201 09S E GC/MS, equ ipp ed wi th a Rtx 95 MS capill ar y colum n (R est ek , Bell efont e, C A – USA). The ch rom ato grams an d m ass spect ra were ev al uated usin g th e GCMSol utio n so ft ware. Co mpo und s were ind enfied b y t hei r mass sp ect ra and ret ention ti me in dex mat chin g to th e mass sp ectral coll ectio n o f t h e NIS T11 dat ab as e. Numeri cal anal ys es were b as ed o n th e p eak hei gh t v alu es o f t he reco rd ed m ass featu re. Th es e val ues were co rrect ed for th e dr y wei ght o f each samp le and b y th e res pons e o f th e i nt ernal stan dard from each resp ectiv e GC 9 MS chromato gram to obt ain no rm alized resp ons es .
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The chan ges i n th e molecular o rgan ic com pou nds of th e MAOM fractio n were as ses sed usin g o ff9li ne tet ram et h yl ammo ni um h yd rox id e
9
(TM AH) m edi at ed t hermo ch emo l ysis (d el Rio et al ., 19 98 ). Th e TM AH9 thermo ch em ol ysi s, pro du cts were an al yz ed b y gas ch rom ato grap h y9mass spect rom et r y in a Sh imadzu QP 2 010 9SE GC9MS equ ipp ed wit h a Rtx – 5MS col umn (30 m l en gth; 0.25 mm ID; 0.2 5 µ m fi lm t hi ckn es s). Ul trapu re He was used as t he carri er gas at a fl ow rat e o f 3 m L min9 1, th e ion sou rce t emp erat ure set to 200 °C, th e i nt erface tem p eratu re to 290 °C an d o v en t em peratu re ramp from 6 0 °C to 30 0 °C at a rat e of 15 °C min9 1. Th e el uted co mpoun ds in t he ch rom ato grams were id enti fi ed usi n g th e N IST 20 11 mass sp ectral lib rar y and ex t ernal st an dards.
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In ord er to ass ess po ssibl e ch an ges i n t he soil mineralo g y an d th erm al resist en ce o f th e S OM to d egrad atio n, s ynch rot ro n XR D an al ys is coupl ed t o Evol ved Gas An al ys i s throu gh m ass s p ect rom et r y (EGA9MS ) was p erfo rm ed in th e Brazili an S yn ch rot ron Li ght So urce. Briefl y, 1 00 m g of t he MAOM fractio n o f each treat ment and from th e 093 and 3 96 mm di st an ces were h eat ed whil e XR D an al ys is was p erfo rm ed. Thi s was acompli sh ed b y couplin g an “Anton Paar XRK900 ” fu rn ace to th e b eam en d s tat ion and heati n g t he s ampl e from room t emp erat u re up to 5 00 °C , und er an ai r flow o f 15 0 m L/min (80 % He, 20 % O2) at a h eatin g rat e o f 5° C/m in . Th e evolv ed gases were sampl ed and an al yz ed t hrou gh out th e cou rs e o f th e whol e h eatin g pro ces s b y a co upl ed mass sp ectrom eter (QMA 20 0 9 Pfei ffer Vacum ). Th e m onit o red m/z v al ues were 17 and 18 (OH and H2O), 2 7 (HCN), 28 (CO), 30 (NO), 4 4 (C O2 and
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N2O) and 4 6 (NO2).The XRD pattern s were cons tantl y t aken b etween 10 an d 50 2 θ d egrees th ro u gh th e who le tim e th e therm al anal ys is was carri ed out .
The EGA9MS d at a fo r H2O and C O2 were an al yz ed u sin g th e s oft ware Peakfi t b y adju stin g expon enti all y mo d ifi ed Gaus sian fu nct ions for si gnal decon vol utio n and area cal cul atio n. Th e XRD pat terns were anal yz ed usi n g the XP owd er so ftware fo r p eak id ent ifi catio n, p eaks FWM H (full width at peak h alf m ax imum) of sel ect ed cl a y min erals and phas e ch an ge th rou gh t he heatin g p ro cess .
The ass umpti ons for hom o gen eit y o f varian ces and n orm ali t y were test ed fo r each s am ple b y Lev en e’s t es t and Kolmo goro ff9Smirn off test resp ectiv el y. Di fferences in t he ab ov e ground m at erial , th e root d r y m ass , root trait s and C O2 concen trati ons were t est ed usin g con fid en ce int ervals (p < 0.0 5). Ch an ges in th e s oils p h ysi co 9chemi cal ch aract eristi cs were t est ed usin g a two9wa y ANOVA foll owed b y Dunn ett 9test (p < 0.0 5 ). All an al ys es were p erform ed in th e STAT IST IC A s oft ware (Statso ft In c.).
(
The N d efi ci en c y in the abo vegroun d m at eri al h eavil y affected th e ass ess ed charact erist ics (Tabl e 1 ). Th e new abov egro und m ass i n th e +N treatm ent was h i gh er than th at in t he –N t reatment. Wh en an al yzed th e l eav es
11
thro u gh th e SPAD in dex , a hi gher N leaf cont ent in th e +N t reat ment s agai nst that o f th e –N treatm ent was obs erv ed.
The root m as s grown in th e lo wer comp artment box was eq ual fo r bo th N t reatm ent s. Sti ll th e Ro ot:Sh oot ratio was s i gni fi cantl y hi gh er for th e p lants of th e –N t reatm en t than fo r t h e +N t reat ment . Th e m ai n ro ot trait ass ess ed thro u gh th e G IAro ot s so ft ware was t he su rface area, whi ch was hi gh er in th e –N treatm ent th an in th e +N t reatm ent (Tabl e 2). Thi s was expect ed as t he root s of t he 9N t reat ment were hi ghl y b ranch ed an d d ev elop ed to s econ d and third o rd er. Th e roo t s from th e +N treatm ent i nst ead were less bran ch ed an d thicker (S upp lem en t ar y Fi gu re 4). As expect ed, m ore b ran ch ed root s led to hi gh er su rface area t han l ess bran ched ro ots.
A prelim in ar y ev al uation o f th e met aboli c p ro filing sh owed hi gher and equ al co n centrati ons of cit ri c acid an d o x alic acid resp ectiv el y, in th e root tissu e of th e N9un fertilized t reatm ent (Fi g. 1). Amon g t h e s u gars ex am in ed lower con cent ratio ns o f su cros e an d fru ctose were obs erv ed i n th e root s of the N9un fertil ized pl ants . Still hi gh er con cent rations of tall os e were foun d in the in th e ro ots o f th e N9un fertil ized pl ants . S ev eral am ino aci ds were ex amin ed , an d in al l cases th e roots o f th e N9fertilized p lants s howed hi gher con cent ratio ns.
The con cent ratio n of CO2 in th e soil wit h in t he n o p lant co ntrols (+N & 9N) and t he +N pl ant ed treatm ent were simil ar. However, t he –N pl ant ed treatm ent att ain ed si gnificant l y hi gh er C O2 con centrati ons (Fig. 2 ).
12
%
The unpl an ted +N t reatm ent pres ent ed n o di fferences from th e unpl ant ed 9N t reat ment , si gni f yi n g t h at n o ch an ges wit hin t he lo wer com partm ent are du e direct N ad ditio n (Fi g. 3 ). Th e MAOM fractio n su ffered ch an ges from th e cul tivat ion with Eu cal yptus bot h +N an d 9N. Th e C co nt ent of t he MAOM fracti on d ecreas ed fo r b ot h pl ant ed (9N an d +N) t reatm ents withi n th e fi rst 3 mm wh en comp ared to t he n o pl ant co ntrols. This d ecreas e onl y contin ued fo r t h e p lanted –N t reatment up to 6 mm fro m t he ro ot p lane.
An in creas e o f t he MAOM δ1 3C o f bot h pl ant ed t reat ments (+N and 9 N) in com paris on to th e cont rol was obs erved. The ex tract ed soi l s olut ion pres ent ed a simi lar h ad hi gh er co ncen t rat ion s o f cit rat e i n bot h p lanted treatm ents t han in th e unpl an ted con tro ls. This di fference between t he pl ant ed treatm ents and th e con tro l d ecreased al on g th e ax is. Th e chan ges in th e MAOM tot al N co n tent were v er y cont rastin g fo r th e pl an ted t reatm ents withi n th e fi rst 3 mm, in creasi n g t o 5 % fo r –N plant ed treatment and decreasi n g b y 7 % in th e +N pl ant ed on e.
Chan ges in ex tract able Fe an d Al fro m the MAOM fract ion were ass ess ed th rou gh t h e ox alat e9citrat e m eth o d. These d ecreased for Fe and onl y in th e dist an ce up t o 3 mm fo r th e –N pl ant ed t reatment, relative t o th e no plant cont rol . No si gnifi cant ch an ge was obs erved in ox alate ex tract ab le Al, fo r bot h plant ed t reatment s (+N an d –N) rel ati ve t o bot h co nt rols (+N and – N).
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Chan ges in pH i n bo th pl ant ed t reat ment s of up to 0.6 pH u nits l ess than th at o f th e u npl anted cont rol were obs erved. This val u es were mai nt ain ed withi n th e wh ol e 1 5 mm of t he –N pl ant ed t reat ment. Th e +N pl ant ed treatm ent, ho wever, had a so il p H decrease onl y wi thin th e fi rst 3 mm.
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Kaolinit e, gibb sit e and go ethit e were t he m ain cla y m in erals fou nd in the MAOM fract ion of th e soil we us ed. Th e m ain featu res o f Kaolinit e, Gibb sit e and Goethit e were i nv esti gat ed thro u gh s ynch rot ro n XRD at room temp erature, an d are sum marized in th e Tab le 3 . No ch an ges were s een in neit her th e d sp acin g o r FWM H of Kaolin ite o r Gib bsit e’s mai n p eaks i n an y treatm ent. Ho wev er, chan ges in d s paci n g an d FWM H are observ ed in th e Go ethit e p eak o f th e –N pl ant ed treatm ent , goin g from 0.8 26 for th e con trols to 0.531 . The d9sp aci n g o f goet hit e’s 1 01 hkl i n th e s am e t reat ment , h ad als o an sli ght ch an ge goi n g from 4 .15 to 4.13.
Chan ges in C O2 ev olutio n from t h e th erm al deco mpos itio n of t he MAOM fractio n, wit hin t he t emp erat ures ran ges o f 155 , 320 and 5 00 °C are summ ariz ed in Tabl e 3. At th e fi rst 3 mm, t he M AOM fractio n from bot h planted t reatm en ts (+N and –N) h ad a similar decreas e of CO2 evolut ion com pared to th e unp l ant ed cont rol s. Nev erth el ess , the M AOM of th e pl ant ed –N t reat ment h ad a mu ch st ron ger d ecreas e o f C O2 ev olutio n in th e temp erature ran gi n g of up to 500 °C, wit hin th e s am e 3 mm. At th e s econ d dist an ce (3 – 6 mm), the +N plant ed treat ment i ncreased its C O2 evo luti on b y
14
up to 14 % with in t h e first 1 55 °C , and d ecreas ed it b y 9% wi thin t he 32 0° C ran ge, in relation to the –N unpl ant ed co ntro l.
The CO2 evol utio n p att ern from th e MAOM fracti on o f th e –N planted treatm ent wi thin th e secon d di stance had a simi lar b ehavio r to that of th e first dist an ce (0 – 3). Neverth el ess , th e d ecreas e in C O2 ev oluti on from th e fi rst 155 ° C was l ess p ron oun ced t han t hat in th e fi rst 3 mm . No si gni ficant ch an ge was obs erved i n th e CO2 evol utio n withi n th e 50 0 °C ran ge for bot h plant ed treatm ents at th e s econd dist an ce.
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At th e t ermin atio n of th e stud y, we perform ed an o ff9lin e TMAH9 GC/MS t o ex ami ne t he mol ecul ar comp o sition o f o rganic co mpoun ds in t h e MAOM fracti on s ep arat el y from so ils collect ed at different dist an ces from the root pl an e (Fi g 4 ). Th e m ol ecul ar charact erizati on of th e com pou nds in the unpl anted t reat ment s p rov id ed con trols to com p are ch an ges in S OM ch emist r y fol lowin g the rhizo sph ere p rimin g. A d etailed tabl e o f all identified comp oun d s in each treatm ent at each dis tance can b e viewed i n th e suppl em ent ar y m aterial (S upp . Tables 4, 5 and 6 ). At t he n o p lant cont ro ls, a rat h er hi gh abun dan ce o f al iph ati c comp ound s was obs erv ed, to get her wit h sub eri n d eriv ed and N9bearin g co mpo und s. Th es e relat iv e abu ndances were con sist ent alon g th e whol e ax is. Th e ali phatic compou nds were alwa ys th e domin ant gro up in all s ampl es . At th e cl osest dist an ce, an accum ulat ion o f N9bearin g compo und s an d a d ecrease of alip hat ic co mpo und s was ob serv ed in th e M AOM fracti on o f th e –N pl anted treatm ent and th e oppo sit e effect
15
(d ecrease o f N9b eari n g and in creas e o f aliph ati c comp oun ds ) in t he s ame fractio n o f t he +N pl ant ed treatm ent .
At th e s econd dist an ce, th e p ropo rtio nal d ifferen ce b etween th e con tro ls and th e –N planted treatm ent t reatm ent st arted to fad e awa y. Still a hi gh er num b er of al iph atic comp oun ds were id ent ifi ed for t he –N plant ed treatm ent an d a hi gh er p ropo rtion o f N9b eari n g compo und s were foun d i n t he +N pl ant ed treatm ent. Alon g t he wh ol e ax is in all sampl es no relativ e abu nd an ce di fferen ces fo r th e cuti n an d suberi n d eriv ed compou nds was obs erved.
(
We ev al uated th e effect o f ) rhizod ep ositio n on mineral
boun d SOM b y em pl o yin g th e rhizob ox sys tem d ev eloped b y Wenzel et al ., (20 01 ). Th is s yst em allo wed us to b oth cultiv at e plants u nd er opt imal N and oth er n utri en ts sup p l y an d with out N s uppl y v ia th e upp er comp artm ent, hav in g th e adv ant age o f n o direct int erference from nut ri ent add ition wit h t he rhizos ph eric s oil in t he lo wer comp artm ent. It also en abl ed u s to ass ess t he effects o f onl y rh izo dep ositi on, with out t he p h ysi cal effect s o f ro ots o n soil (eg. di srupti on o f aggregat es, C an d N co ntri butio n o f d ead ro ots, et c). Th e memb ran e’s m esh (5 µm) guarant eed t hat no root h air cam e in direct cont act with th e s oil, thu s s afek eepi n g t h at th e effects of th e pl ant ed treatm ents on the soil are o nl y res u lt o f pl an t rhizod ep o sition .
16
The nit ro gen st atus affects pl ant s in a great mann er, as N i s requi red to s yn th etiz e amin o acids, n ucl eo tid es an d s econd ar y met abol ites th at have direct rol es in pl an t si gn ali n g, struct ure and ad apt ati on (Tsch oep et al., 2009 ). Thu s, it was n o surprise t h at, in t he cu rrent experim ent a great er sh oot dr y m ass was p ro du ced b y +N pl an ted t reatm ents . P h ysi olo gical chan ges in the p lant du e th e l ack of N led to sm all er and yel low l eav es i n the –N pl ant ed treatm ents. This resp ons e is di rectl y rel at ed t o the lack o f N, as it is necess ar y fo r chl oroph yl l s yn t hesi s, l eadin g t o l ower C O2 fix atio n and thus l ess C for stru ct ural and ph ys io lo gi cal fun cti ons (Hermans et al., 2 006 ). This im balance led to imp ort ant ch an ges in th e so urce/d rai n fun ctions o f C and N i n t he pl an t, as i t can b e obs erv ed b y t h e m et aboli c profili n g data (Fi g 1 )
The rhizob ox des i gn all owed us t o effi ci entl y ass ess root m o rph olo g y and ch aracteris tics. This b ecau se th e remov al o f t he root s in th e lower com partm ent was qu ick and with out cau sin g no tabl e dam age. Ou r resul ts show t hat wh en l ack in g N, p lants in vested C to d ev el op thi nn er and l on ger root s (Tabl e 2). Th ese t hin ner ro ots will in t urn ex udate mo re and h av e a larger s urface area (Os ton en et al., 20 0 7) optimizin g nut ri ent upt ak e and increasin g its effect on th e adj acent so il. Ou r resul ts are line with th e obs ervat ion s s ho wn b y M arschn er et al., (1 996 ) and b y Herm ans et al., (20 06 ), who sho wed t hat un der N defi ci en c y p lant partiti on of photo as simil at es ch an ged, redi rect in g mo re C t o its roots .
Ou r root m et ab oli c pro fil e o f t he –N pl ant ed treatment, sho wed no accumul atio n o f su crose in th e ro ot ti ssu e, but o f o th er su gars lik e tall os e.
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This in tu rn m a y hav e regul at ed gen e exp ressi on lead in g to mo re cell divi sion and elon gation (Ko ch, 200 4). No accum ulati on o f amin o acids i n t he –N planted t reatm en ts was obs erv ed, wh en co mpared t o th e +N pl anted treatm ent . We h yp ot hesize th at it was poss ibl y to a preferential accum ulati on in th e leav es.
Ou r resu lts o f root d r y m ass i n th e lo wer box are i n lin e with t hos e b y J ourd an et al., (2 008 ), who i n a fi el d exp eri ment sh owed th at in th e abs en ce
of N, ) pro du ces a great er root m as s th an wh en fertiliz ed with
N. Ev en tho u gh ou r ro ot dr y m ass was th e s am e fo r both t reatm en ts t he root :sho ot rati o in creased un der N defi ci en c y, t his con curs with th e b arl e y dat a from Farrar et al., (200 3), th us imp l yi n g a high er C inv estment in roo t fo rmation .
(
The pl ant s di stin ct N s tat us resul ted in d ifferent effect s on th e rhizos ph eric s oil. A first si gn of t he p lant 9in du ced chan ges in bot h N9 fertiliz ed an d un fert ilized rhizo sph ere s oils is t he differen t soil air C O2 con cent ratio ns. The cont rastin g C O2 v alu es, h i gh er i n t h e un fertiliz ed treatm ent mi ght b e resu lt o f eit h er hi gher respi ration b y t he ro ots o f th e unfertiliz ed treatm en t, d ue to a hi gh er C t ranslo catio n t o t h e root an d hi gh er activit y (Mars ch ner et al., 19 96 ). M ore l ikel y, h owev er, t he greater s oil ai r
CO2 is th e result of a pl ant 9roo t ind uced SOM d ecomp ositi on b y th e h i gh er
18
As we fu rth er an al yzed di fferent ph ys ico9ch emi cal p aram et ers in th e soils it b ecam e app arent t hat di fferent m ech anis m for primin g were in actio n dep en din g o n th e N stat us o f t h e pl ant s (Fi g. 3 ). A hi gher C mineralizat ion in bo th t reatments (Fi g. 3 a) i ndi cates th at ev en at opti mal N s tat us m a y indu ce a pri min g effect at th e clo sest di stan ces to th e root . We b eli ev e th at th is p rimi n g was caus ed b y t he opt imal ex ud at e com positi on in th e N9ferti lized t reatm ent. Si nce there was no im balance of su gars, amin oaci ds and o rgani c acids i n t he N9fert ilized roots , the sam e m a y be tru e for its ex ud ate. Th us, th e i ncreas ed C min eralizati on ma y b e d ue to microbi al stoi ch iom etri c con strai nts. Thi s , is in li ne wi th t he wo rk of Drak e et al., (20 13 ), were C9N b al an ced arti fici al ex ud ate so luti on in du ced a positi ve p rimi n g b y p rovi din g N ri ch s ubst rate fo r t he mi croo rgani sm to s ynth etiz e ex oenz ym es
Altho u gh t he ch an ges in C withi n th e fi rst d ist an ce do not di ffer fo r both treatm ent s, in t he s econd di stan ce (3 – 6 mm ) a hi gher m ineralizati on in the un fertilized t reat ment was obs erved, similar b eh avi or p resent ed t he p H and Feox . As hi gh er cit rat e con cent ratio n s were fo und i n th e N9un fertiliz ed root s and M AOM fracti on (Fi g 3 . c), we b eli eve t hat t he u nfertiliz ed ex ud at ed com pou nds with m ore o f a com pl ex in g b ehav ior in o rd er to h av e acces s to th e N i n t he min eral bo und organic m att er. As revi ewed b y Fu rrer and S tumm, (1986 ) and Zin der et al., (198 6), ci trat e and ox al at e are impo rtant Fe an d Al com plex ants wit h hi gh di ssol utio n rat es . The acti vit y o f such o rgani c compl ex ants in t he un fertili zed t reatment was confi rm ed b y a
19
decreas e i n Feox (Fi g 2 d), and b y shi ft s in t h e d 9sp acin g and FWHM o f goethit e ( Tabl e 3 and 4), whil e at th e fertiliz ed t reatm ent no s i gni fi cant ch an ges were ob serv ed.
The N cont ent at t he MAOM fracti on o f both pl anted treatm ents h ad con trasti n g v alu es (Fi g.3 b), as th e –N planted t reat ment i n creas ed and t h e +N p lanted treatm ent decreas ed . Thi s led u s to b eliev e t hat a di fferent microbi al d yn am ic acted as a driv er for eit her th e accum ulati on or th e dep leti on o f N n ear t he ro ots. Pl ants ch an ge its exud at e com p ositio n d ue to nutrient st atus (C arv alh ais et al., 201 1), and th at wa y benefit/ affect di fferent microbi al comm uniti es. As Dij kst ra et al., (2 013 ) exp ress ed , as a easil y met abol izab le C is mad e avai lable for m icro organisms , opp ort unis tic comm uniti es (r9strat egis t) ma y ros e to h igh er pop ulation s th an t hat o f th e nati ve slo w growt h communit ies (K9st rategi st).
The pl ant s u nd er N defi ci en c y tri ed to sup pl y t h ems elv es with N thro u gh oth er m ech anisms lik e hi gh er roo t b ran chin g and hi gh er o rgan ic acid ex udation . Du e its N defi ci en c y, we b eli ev e t h at sugars and N9b earin g com pou nds were in s tead accumu lat ed in th e l eav es as it h as al read y b een obs erved in oth er pl ants un der N d eficienc y (P aul and Dri scoll, 1 997 ) an d this can act as si gnal for ro ot el on gati on and growt h (Herm an s et al., 2 006 ). To geth er wit h th e o rgan ic acid ex ud atio n, a rat her hi gh ch an ge in soil pH was obs erved (Fi g.2 f), whi ch m a y h av e b een t he co ns equen ce o f th e hi gh C O2 con cent ratio n o r du e th e pl an t p rot on ex trusi on. Eit her wa y, t hes e ch an ges
20
ma y h av e furth ered t he di ssol utio n of Fe ox y(h yd ro)x id es b y l oweri n g th e Eh and p H o f t he so lutio n.
The un ferti lized pl an ts primin g mechanis m mad e mi neral b ou nd SOM av ail abl e fo r t he mi crobi al commu niti es. The hi gh er C av ail ab ilit y l ead to a microbi al pop ul atio n increase, sp eci fi call y t h e r9st rat egis ts as these are th e ones b ett er p rep ared fo r n ati ve OM min eralization (P hilip pot et al ., 201 3).
Differen ces in th e mi neralized C an d N at the vi cinit y o f th e ro ots was also con fi rmed b y EGA and TM AH9 GC/ MS (Tabl e 3 and Fi g. 4 ). Th e EGA9 MS CO2 evol utio n s howed rat her hi gh C di fferen ces bet ween th e MAOM fractio n from th e fertiliz ed and th e un fert ilized treatm ent s withi n th e C fractio n th erm all y resist ant fractio n (between 30 0 and 50 0 °C). Th e C O2 deriv ed from th e M AOM fractio n co ntin u ed to differ at th e secon d d ist an ce, ex cept th at th ere were also lo wer areas withi n t he mo re th ermall y l abil e C fractio ns (15 5 – 30 0 °C). We beli eve th at this di fferen ces are d ue ch an ges in the chem ical compo s ition of th e mi neral boun d S OM fractio n.
The TM AH9GC/MS dat a co nfi rm ed wh at the bul k an al ysis s ho wed fo r the un ferti lized treat ment , an i ncreas e o f N9bearing comp oun d s in t h e vi cin it y of t he root pl an e an d a d ecreas e i n alip hati c com pou nds , whi ch cou ld explain the m ain ch an ges in t he p att ern o f CO2 evolu tion from th erm all y decom posit ion o f th e MAOM fraction o f S OM. Alth ou gh in bo th pl ant ed treatm ents (+N and –N) t h e C decrease was t h e same, the m olecu lar con stitut ion o f each was different, as th e MAOM fracti on fro m the fertiliz ed
21
and pl ant ed treatm en t had a m uch hi gh er cont ent of al iph ati c co mpoun ds t h an ev en th e n o pl ant co ntro ls.
We bel ieve t hat s uch cont rastin g effects i n S OM chemist r y am on g th e treatm ents was du e to a different ial res pons e of the dom in ant mi crobi al comm uniti es an d cau sed b y th e v ariabl e compos ition of ex ud ates. Alth ou gh root ex ud ation is in dep en d ent o f t he N9s tatu s, th e ch emi cal compos ition o f the ex ud at es is bo und ed to th e plant N st atus . Thu s, t he mi crobi al comm uniti es b en efit ed are cons equ en ce o f th e pl ants nu trit ion al necessit ies.
%
In th is experim ent, we had anti cip at ed th at th e s oil u n der N9 unfertiliz ed pl ants would giv e a hi gher S OM min eralizati on d ue t o a posi tiv e primi n g effect . Alth ou gh th is was true, we di d not expect t o h av e also a positi ve p rimi n g effect i n t h e N9fertilized t reatm ent. Th is led u s t o believe that ev en th ou gh in b oth N t reatm ent s th e plants ind uced S OM mineralizat ion , different m ech ani sm s of p rimi n g were un der effect , whi ch affected th e SOM ch emist r y in di fferen t wa ys .
While both pl ant ed treatments i ncreased C min eralizati on, the N9 defi cient pl ants affected a hi gh er rhizos ph eri c soil v olum e. Thi s l arger effect was du e a com bin ati on o f ch an ges in p h ysico ch emi cal fact ors (soi l p H, C O2 con cent ratio n) and a hi gh er ex udat ion o f organi c acid s.
The Fi gure 5 (m od ifi ed from (Ch en et al., 2 014 )) ill ust rat es th e domin ant pri min g m ech anis m un dergoin g at each planted t reat ment . Th e d at a
22
indi cat es t hat , ex ud at e com posi tion is i nflu en ced b y t h e pl ants N status . Und er N9d efi cien c y plants ex ud at e mo re organi c acids and l ess en erg y ri ch com pou nds, thu s p rev ailin g th e “N9m i nin g” p rimi n g m echan ism. Wh en suppl ied wit h N, th e plants rel eas e a mo re com pou nd di vers e ex udat e, l eadin g to t he d omin an ce o f t he “microbi al stoi ch i omet r y” mechanism . The different domin ant m ech ani s ms indi cate t h at di fferent mi crobial communit ies are ben efit ed d ep end in g on t he pl ant N status . As t he N9defi ci ent plants affect ed more t he ph ys i co ch emical ch aract eristi cs of t he so il, an d m a y hav e in creas ed the p opu lat ion of K9strat egist s, a global scen ario in whi ch pl ants und er N defi cienc y i ndu ce th e mi neralization o f mineral associat ed C is p lausibl e.
These res ults are t o o ur k no wl ed ge, t h e fi rst d emon strati on o f d ifferent primi n g m echanisms due t o plant N9st atu s . Ou r wo rk , refl ects t he imp ort ance of careful nut ri ent man agem ent i n fo rest plant atio ns, and hi ghli ghts t he possi bilit y of hi gher C mineralization i n N9defi ci ent fo rests. We recomm en d that futu re stu di es, t ak e i nto accou nt ot h er imp ortan t n utri en t s su ch as P, K.
23
M ean Leaf9SPAD Index, new sh oot and root d r y m ass and ro ot:sh oot rati o of Eucal yp t s eedli n gs as affect ed b y N s uppl y. M eans foll owed b y th e s am e case l ett er do not di ffer b y F9t est (p <0.0 5 ).
Parameter
+ N 9 N
Shoot Root Shoot Root
Leaf9SPAD Index 41.33±1.76 a 9 27.33±1.20 b 9
Dry mass (g) 2.01±0.14 a 0.42±0.04 A 1.19±0.04 b 0.41±0.02 A
Root:Shoot 0.21±0.02 b 0.35±0.05 a
+N is the fertilized treatment and -N the unfertilized one.
M ean root t rai ts estim at ed at th e end of t he exp erim ental perio d for Eucal yp t s eedli n g s und er distin ct N9suppl y comp ut ed b y the GiaR oots Soft ware. Mean s fol lowed b y t h e s am e case l ett er do not di ffer b y F9test (p <0.05 ).
+N is the fertilized treatment and -N the unfertilized one.
Root traits + N 9 N p9value
Root average width (mm) 3.77 A 2.84 A 0.087
Root length (cm) 900.64 B 1814.34 A 0.049
Root surface area (cm2)
24 ! ! " ! # $ % ! & !' ( ) ) ) ) ) ) ) )
Metabolic profiling of the lower compartment roots from – N planted treatment. The dashed line indicates the relative abundance of each compound in the +N planted treatment. Values differing from the +N planted treatment denote a treatment induced change, signed by an asterisk (F9test, p < 0.05). Values are shown as relative abundance of each compound to the same compound in the +N planted treatment ± s.e.m. (n=3).
' ' $ * + , * - ' - ' - ' - ' δ 1 3 . * + ,
(a) Mean soil CO2 concentrations and (b) δ13C9CO2 of the rhizospheric soil air, 16 hours after a pulse with 13C enriched CO2 at the end of the experimental period. Values differing from the control (9Pl/9N) denote a treatment induced change, signed by an asterisk (F9test, p < 0.05). Values are shown as means ± s.e.m. (n=3).
25 a) b) c) d) e) f) g) / -0 ( 1 ' - / -0 ( 0 ( " 0 ( 0 ( ! 2 $ 0 ' $$ + + ' 3 0 ( 4 4 4 4 4 ! 2 $ 0 ' $$ + + ! 2 $ 0 ' $$ + + ) ) ) ) ) ) ) ) ) ) ) ) ) δ 1 3 0 ( . 4 4 4 4 4 ) ) )
Induced effects of ) exudation on the MAOM’s C, N, δ13C and soil
solution Citrate concentration. (a, b, f and c), protective mineral phases and in soil pH (d, e and g), presented as function of distance from the root plane. Effects were calculated as the
26
percentage difference between concentrations in the treatment and the control (9N unplanted treatment) or as diference of the treatment to the control value. Values differing from that of the dashed line denote a treatment induced change, asterisks sign a significant change from the control (two way ANOVA, followed by Dunnet’s test p < 0.05). Values are shown as means ± s.e.m. (n=3).
Relative changes in CO2 (m/z 44) evolution from the MAOM fraction from plants fertilized (+N) and not (9N) with Nitrogen. Percentages are relative to the CO2 evolution of the no plant control.
Temperature Range °C
+Pl/+N +Pl/-N +Pl/+N +Pl/-N
Distance from the Root Plane (mm)
0 - 3 3 - 6
0 - 155 -32% -37% 14% -16%
155 - 320 -15% -13% -9% -19%
320 - 500 -3% -24% 6% 0%
27
. Changes in the goethite mineralogy assessed by synchrotron based XRD.
Treatment Mineral hkl d (Å) FWHM -Pl/-N Goethite 1 0 1 4.1580 0.826 +Pl/-N 4.1314 0.531 -Pl/+N 4.1530 0.826 +Pl/+N 4.1492 0.732
+Pl is the planted treatment, -Pl the unplanted one, +N the fertilized treatment and -N the unfertilized one. FWHM: Full width at half maximum
28 0 – 3 mm 3 – 6 mm 6 – 15 mm * + , * + , '0 $ & ( - ( / 5 - / 5 - / 5 - / 5 -* + ,
Relative abundance of chemical compound groups in the mineral associated organic matter fraction in the distance 0 – 3, 3 – 6 and 6 – 1.5 mm from the root plane. +P & 9P denotes the planted and unplanted treatments respectively. +N & 9N denote the fertilized and unfertilized treatments.
29
Possible dominant mechanisms of nutrient9driven priming effect in the planted treatments under N deficiency and high N availability. The blue lines show fluxes controlled by native microbial populations (K9strategists) and red lines show fluxes controlled by opportunistic microbial communities (r9strategists). Adapted from (Chen et al., 2014).
30
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!
Suppl em ent ar y Fi gu re 1 . Rh izobox d esi gn an d cross sect ions . Legen d: (1 ) soil–plant com partm ent; (2) tran sp aren t acr yl i c wall; (3 ) n yl o n m emb rane (mesh widt h 5 µm); (4) rhizo sph ere so il comp artm en t; (5 ) i rri gati on wi cks (fi ber gl ass, Fib ers eals, d iam et er 5mm ).
38
Suppl em ent ar y Table 1. Modi fi ed Clark S olutio n u sed fo r th e rhizobox experim ent . Macronutrients (mmol/L) Ca K N9NH4 N9NO3 Mg S P 2.6 1.8 3.5 3.5 0.6 0.6 0.138 15N9NH 4 0.01 Micronutrients (mmol/L) Fe B Zn Cu Mn Mo 0.09 0.019 0.02 0.0005 0.007 0.0006 Others Al 0.05
Suppl em ent ar y Fi gu re 2 . Soi l9ai r gas s am pler. Th e fl at s ili con e coil is fix ed with tape (not sho wn ) to m aint ain th e fl at fo rm. The end of th e si licon e coil was clos ed wit h a pl asti c shutt er.
39
Suppl em ent ar y Table 2. Two9wa y ANOVA results for ph ys i co 9chemical properti es o f th e MAOM fractio n (tot al organ ic C, δ13C, Fe9oxalate and Al9 oxalate and total N), Citrate concentration in the soil solution and soil pHH2O
Distance Treatment TOC δ13C pH Fe-Ox Al-Ox Citrate N p Value 0 - 3 Nitrogen 0.01 0.77 4 4 4 6 0.00 0.00 Plant 0.00 0.01 4 4 4 6 0.00 0.49 Plant x Nitrogen 0.47 0.76 4 4 4 0.00 0.00 3 - 6 Nitrogen 46 4 6 4 4,* 4 , 0.00 4 Plant 4 4 6 4 46 4 0.00 4 Plant x Nitrogen 4 4 4 46 4, 0.01 4 6 - 15 Nitrogen 4 4 , 4 4 + 4 0.00 0.32 Plant 46, 4 4 4 6 46+ 0.00 0.98 Plant x Nitrogen 4 * 4 4 4+7 4 7 0.00 0.54
40
Suppl em ent ar y Fi gu re 3 . C rop ped an d th resh old ed image o f an (a) N9 unfertiliz ed t reatmen t and a N9fertilized t reatm en t, after data t reatm en t in the Gi aRo ots so ftware.
a)
41
Supplementary Figure 4. CO2 (m/z 44) and H2O (m/z 18) evolution of the MAOM fraction along the heat ramp during thermal decomposition.
8 $ $ ! 8 $ ' 9 * + % 4 4 4* 4+ 4, 4 4 8 $' 3
42
Suppl ement ar y Fi gure 5 S ynchrotron9based X9ra y di ffraction patt ern evoluti on during t he thermal
decomposit ion of t he MAOM fraction. Main features incl uded are: ( Kaolinit e: 12 °2θ, Gi bbsit e: 18 °2θ, Goethit e: 21 °2θ and Quartz: 27 °2θ).
43
