Transcriptomic and metabolic responses of mycorrhizal roots to nitrogen patches under field conditions

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1 Plnt Soil () 35:5 6 DOI.7/s--89-z REGULAR ARTICLE Trnscriptomic nd metolic responses of mycorrhizl roots to nitrogen ptches under field conditions Dniel R. Ruzick & Ntsh T. Husmnn & Felipe H. Brrios-Msis & Louise E. Jckson & Dniel P. Schchtmn Received: 3 April /Accepted: June /Pulished online: 3 July # Springer Science+Business Medi B.V. Astrct Bckground Arusculr mycorrhizl (AM) fungi contriute to plnt nutrient uptke in systems mnged with reduced fertilizer nd pesticide inputs such s orgnic griculture y extending the effective size of the rhizosphere nd delivering minerls to the root. Connecting the Responsile Editor: Peter Christie. Dniel R. Ruzick nd Ntsh T. Husmnn contriuted eqully to this work. Electronic supplementry mteril The online version of this rticle (doi:.7/s--89-z) contins supplementry mteril, which is ville to uthorized users. D. R. Ruzick : D. P. Schchtmn (*) Donld Dnforth Plnt Science Center, 975 N Wrson Rd., St. Louis, MO 633, USA e-mil: dschchtmn@dnforthcenter.org D. R. Ruzick e-mil: druzick@dnforthcenter.org N. T. Husmnn : F. H. Brrios-Msis : L. E. Jckson Deprtment of Lnd, Air, nd Wter Resources, University of Cliforni, Shields Avenue, Dvis, CA 9566, USA N. T. Husmnn e-mil: nteutsch@gmil.com F. H. Brrios-Msis e-mil: frrios@ucdvis.edu L. E. Jckson e-mil: lejckson@ucdvis.edu moleculr study of the AM symiosis with griculturllynd ecologiclly-relevnt field environments remins chllenge nd is lrgely unexplored reserch topic. Methods This study utilized cross-disciplinry pproch to exmine the trnscriptionl, metolic, nd physiologicl responses of tomto (Solnum lycopersicum) AM roots to loclized ptch of nitrogen (N). A wild-type mycorrhizl tomto nd closely-relted non-mycorrhizl mutnt were grown t n orgnic frm in soil tht contined n ctive AM extrrdicl hyphl network nd soil microe community. Results The mjority of genes regulted y upon enrichment of nitrogen were similrly expressed in mycorrhizl nd non-mycorrhizl roots, suggesting tht the primry response to n enriched N ptch is medited y mycorrhiz-independent root processes. However where inorgnic N concentrtions in the soil were low, differentil regultion of key tomto N trnsport nd ssimiltion genes indicte trnscriptome shift towrds mycorrhizmedited N uptke over direct root supplied N. Furthermore, two novel mycorrhizl-specific tomto mmonium trnsporters were lso found to e regulted under low N conditions. A conceptul model is presented integrting the trnscriptome response to low N nd highlighting the mycorrhizl-specific mmonium trnsporters. Conclusions These results enhnce our understnding of the role of the AM symiosis in sensing nd response to n enriched N ptch, nd demonstrte tht trnscriptome nlyses of complex plnt-microe-soil interctions provide glol snpshot of iologicl processes relevnt to soil processes in orgnic griculture.

2 6 Plnt Soil () 35:5 6 Keywords Mycorrhizl symiosis. Nitrogen metolism. Plnt-microe-soil interction. Root trnscriptome. Tomto Arevitions AM rusculr mycorrhiz N nitrogen P phosphte Pi inorgnic phosphte Zn zinc S sulfur Cu copper PT phosphte trnsporter + NH mmonium NO 3 nitrte AMT mmonium trnsporter NRT nitrte trnsporter PSR phosphte strvtion response Introduction The rusculr mycorrhizl (AM) fungl root symiosis occurs in the mjority of lnd plnts including most crop plnts, nd my ply n importnt role in nutrient uptke in low input environments (Allen nd Shchr-Hill 9; Govindrjulu et l. 5; Jvot et l. 7; Smith nd Red 8). The AM fungi develop network of extr-rdicl mycelium tht extends into the soil, expnding the effective rnge of the root rhizosphere (Miller et l. 995). Inorgnic phosphte (Pi) is the min soil nutrient supplied y the symiosis nd mycorrhizl-specific plnt Pi trnsporters (PTs) re responsile for the mjority of Pi trnsport into mycorrhizl roots (Jvot et l. 7). Much of the trnscriptionl response to the mycorrhizl symiosis is likely consequence of this dditionl mycorrhizl Pi uptke (Ngy et l. 9). Mycorrhizl plnts lso exhiit higher levels of minerl nutrients including sulfur (S), zinc (Zn), nd copper (Cu) (Cvgnro et l. 8; Liuetl. ); however the plnt trnsporters responsile for the uptke of these mycorrhizl-supplied nutrients hve not een identified eyond trnscriptionl studies in greenhouse-grown plnts (Guether et l. 9). Soil nitrogen (N) is not uniformly distriuted nd is often limiting in griculturl systems mking it n importnt nutrient for mycorrhizl-medited uptke. The vried sources of N nd soil processes led to complex dynmic N pools (Burger et l. 5). Ftes of inorgnic N in the soil include direct uptke y plnt roots or AM hyphe, microil immoiliztion or trnsformtion, nd environmentl losses to nitrte (NO 3 ) leching elow the root zone nd gseous emissions. Initil plnt response to ville N in the environment include the regultion of mmonium (NH + ) nd NO 3 trnsporters nd root prolifertion in loclized res with high N content (Grnto nd Rper 989; Hodge ; Remns et l. 6). While most studies hve focused on single form of nitrogen, our work hs shown tht roots lso hve the ility to respond to the incresed vilility of multiple forms of N during the nitrifiction fter dded NH + (Ruzick et l. ). AM fungi help plnts compete for soil N tht my e lost or immoilized y other soil microes. The extrrdicl mycelium tke up orgnic nd inorgnic N from the soil vi mino cid nd NH + trnsporters, trnslocte it to the intrrdicl mycelium s rginine, nd relese N to the plnt in the form of NH + (Cruz et l. 7; Govindrjulu et l. 5; Jin et l. 5; Leigh et l. 9; Tnk nd Yno 5). Recent studies hve identified Medicgo trunctul nd Lotus jponicus mycorrhizl-induced plnt NH + trnsporters tht re likely to trnsport mycorrhizlderived NH + cross the perirusculr memrne to the plnt (Gomez et l. 9; Guether et l. 9; Koe et l. ). It hs een proposed tht this mycorrhizl N-uptke pthwy is similr in some wys to the well chrcterized mycorrhizl Pi uptke pthwy. The identifiction of significnt downstrem effects of the N supplied y mycorrhizl fungi on plnt physiologicl or iochemicl processes re limited (Hilderndt et l. ). Previous work in controlled greenhouse pot study reported strong nd complex tomto root trnscriptionl response to n enriched N ptch (Ruzick et l. ). In the present study, we used the sme system to explore mycorrhizl root responses to n enriched N ptch. The current study ws conducted in n orgnic frm soil in the field where plnts my hve een suject to greter environmentl heterogeneity, where soil nutrient trnsformtions were more complex, nd where ptches of pplied N cptured smller proportion of the totl root system. This is the first field-sed study to exmine root gene expression chnges in response to mycorrhizl fungi. The

3 Plnt Soil () 35:5 6 7 Fig. Digrm of nitrogen ptch experimentl design nd quntifiction of rusculr mycorrhizl coloniztion. Root ingrowth rings were uried elow ech genotype (76R MYC+ or rmc) nd injected with either high 5 NH + -N (65 μg 5 NH + -N g soil), low 5 NH + -N (6.5 μg 5 NH + -N g soil) or wter to simulte nutrient ptch. Ptch roots were hrvested h fter injection. The mycorrhizl wild-type (76R MYC+) nd the non-mycorrhizl mutnt (rmc) hd different coloniztion rtes y stndrd line intersect methods () nd y qpcr nlysis of Glomus intrrdices lkline phosphtse (GiALP) nd rusculr mycorrhizl ß-tuulin (Amß-tuulin) fungl gene expression (c). Asterisks represent significnt difference etween 76R MYC+ nd rmc for ech comprison (***=P<.). The light nd drk grey rs represent 76R MYC+ nd rmc respectively. The reltive expression ws clculted using the ΔΔCT method with LeACT s the reference control to normlize for tomto tissue input, nd the 76R MYC+ group set to use of reduced mycorrhizl mutnt (rmc) tht is closely mtched in growth to its mycorrhizl wildtype tomto (Cvgnro et l. 8) provided the opportunity to conduct this study in n griculturlly relevnt field setting nd voided mny of the confounding fctors introduced y nti-mycorrhizl tretments. Two concentrtions of 5 NH + nd wter control were pplied to loclized soil ptches contining mycorrhizl wild-type or non-mycorrhizl rmc roots (Fig. ). Twenty-four hours fter the NH + tretments, plnts were hrvested to exmine the effects of the AM symiosis on the root responses to the ddition of N in the ptch. We demonstrte tht while the N-enriched ptches hd profound impcts on plnt gene expression, the symiosis resulted in dditionl nd interctive effects. At lower concentrtions of soil inorgnic N, similr to those typiclly found in lower input orgnic griculture (Mder et l. ; Smukler et l. ), AM fungi supplied dditionl N to the roots. In ddition to chnges in nitrogen responses, we found mycorrhizl-enhnced plnt P, Cu, nd S uptke linked to the coordinted mycorrhizl-regultion of plnt Pi, Cu, nd S trnsporters nd downstrem metolism genes. We propose conceptul model of mycorrhizl root N metolism sed on the trnscription profiling dt where the mycorrhizl effect on N uptke nd metolism is more pronounced nd possily promoted under lower N vilility. This model highlights trnscriptionl repression of direct root N uptke pthwys under low N conditions where mycorrhizl-specific tomto mmonium trnsporters LeAMT nd LeAMT5 re expressed. c % coloniztion Reltive Expression (n=3) Mterils nd methods Plnt mteril *** 76R MYC+ *** 76R MYC+ GiALP rmc *** rmc 76R rmc MYC+ AMBtu Two different tomto genotypes (S. lycopersicum L.) were used: the wild-type cv RioGrnde 76R (Peto

4 8 Plnt Soil () 35:5 6 Seeds) tht forms mycorrhizl ssocitions (76R MYC+) nd the rmc mutnt (derived from 76R (Brker et l. 998)). Plnts were germinted s descried in Ruzick et l. (). Eight-week old seedlings were trnsplnted one foot prt t tomto field on n orgniclly mnged frm, Durst Orgnic Growers Inc. in Esprto, Yolo County, Cliforni (field soil properties re presented in Cvgnro et l. (6)). In order to pply nutrients in ptches nd recover the roots tht were directly exposed to the nutrient ddition, root in-growth rings were uried t 7.5 cm elow the soil surfce nd etween the two experimentl plnts in ech plot (see elow). The rings hd n internl dimeter of cm nd were 5. cm tll (totl volume 8 cm 3 ) nd were filled with 57 g of field soil to finl ulk density pproximtely equl to tht of the surrounding soil (. g cm 3 ). The rod ends of the ring were covered with -mm plstic mesh to llow roots to esily grow into the ring. Plnts were wtered regulrly using.9 l hour surfce drip hose with emitters spced 3 cm. Emitters were plced etween plnts, i.e., 5 cm on either side of every plnt nd directly ove the uried ring. No fertilizer ws pplied except for the experimentl N tretments. Experimentl design Ech experimentl plot consisted of two experimentl plnts nd two outer plnts considered s uffers; plnts totl per plot. There were two genotypes (76R MYC+ nd rmc) nd three nutrient tretments (wter, low NH +, nd high NH + ), nd six plot replictes (iologicl reps) for totl of 36 plots. The trnsplnts were grown for five weeks in the field to llow sufficient root mycorrhizl coloniztion s well s root explortion of the uried ring. Tretments were injected t 3 evenly distriuted positions in the ring in -ml liquots, for 6 ml totl volume. For the low NH + tretment, 6.5 μg 5 NH + -N (99 tom percent) per grm of dry soil were dded in the ring (.97 mg 5 NH + -N per ring) which ws -fold increse over mient soil levels of NH + -N. For the high NH + tretment, 65 μg 5 NH -N (99 tom percent) per grm of dry soil were dded in the ring (9.7 mg 5 NH + -N per ring), -fold increse over mient soil levels. Wter ws used s control. Additionl 76R MYC+ nd rmc plots were used to study 5 N uptke over time. These plots were injected with the high NH + or wter control tretment. The youngest, fully emerged leves of the two middle plnts in ech plot were hrvested one, two, five, seven, nd nine dys post-tretment for 5 N isotope nlysis. Hrvest nd smple nlysis Plots were hrvested h fter the rings were injected y cutting the two plnts on either side of the ring t ground level, nd lter seprting the shoot in leves, fruits nd stems. All oveground iomss ws dried, weighed, nd ground for δ 5 N stle isotope nlysis nd nutrient nlysis. Immeditely fter severing the plnts, the uried ring ws crefully excvted to prevent pulling roots out of the ring, nd ll sides shved with rzor lde. Two, 5 5 mg (fresh weight) susmples of roots were promptly removed from the ring, rinsed, pt dry, nd flsh frozen in liquid nitrogen for RNA extrction under minimized/ indirect light. A representtive susmple of soil ws removed from the rings, M KCl dded t liquid: soil rtio of.5:, shken nd the superntnt nlyzed using modifiction of Mirnd et l. - () for NO 3 nd Foster (995) for NH +. Grvimetric soil moisture ws lso determined. The ckground mmonium nd nitrte concentrtions in the soil ws determined y rndomly collecting nine soil smples from two depths ( 5 cm nd 5 5 cm) for ech genotype (36 soil smples totl). From the remining roots, smll susmple of ptch roots ws scored for AM coloniztion t X (McGonigle et l. 99). All remining roots from the ring were dried, weighed, nd ground for δ 5 N isotope nlysis on PDZ Europ ANCA-GSL elementl nlyzer nd PDZ Europ isotope rtio mss spectrometer (Sercon Ltd., Cheshire, UK) t the UC Dvis Stle Isotope Fcility, USA. Leves were nlyzed for totl P, S, Zn, nd Cu using nitric cid/hydrogen peroxide microwve digestion (Meyer nd Keliher 99; Sh nd Miller 99) followed y tomic sorption spectrometry nd inductively coupled plsm tomic emission spectrometry t the UC Dvis DANR ls. Plnt hormone nlysis on iologicl replictes of ech tretment/genotype group ws performed on lyophilized root tissue hrvested from the nutrient ptch (5 mg fresh weight) y LC/ MS/MS on the ABI QTRAP system t the Dnforth Center Proteomics nd Mss Spectrometry

5 Plnt Soil () 35:5 6 9 Fcility (See Protocol in Online Resource for detiled protocol). Microrry nlysis Trnscriptome profiling of ech h-post injection RNA smple ws performed using the Tomto Genome Arry Chip (Affymetrix, Snt Clr, CA, USA) s descried in Ruzick et l. (). The dt were filtered to remove proesets clled sent y the MAS5 summriztion in ll 3 rrys. For ech proeset, which represents the comined expression dt from ll relevnt proe pirs on the chip, the generlized liner model Y ijk =μ+α i +β j +(αβ) ij +ε ijk ws fit. In ech ANOVA, Y ij is the log normlized trnscript level for the i th tretment, the j th genotype, nd the k th replicte, μ is the overll men expression for the proeset, α represents the i th tretment (wter, low nutrient, nd high nutrient), β represents the j th genotype (76R MYC+ nd rmc), nd (αβ) the interction of the i th tretment nd j th genotype. F- tests were used to test the null hypothesis for ech min effect nd the interction effect. We exmined the model for conformtion to the ssumption of normlity of the residuls testing the null hypothesis tht the residuls for ech gene were normlly distriuted using the Shpiro-Wilkes Test. All nlyses were performed in JMP Genomics. (SAS Institute, Cry NC). An FDR level of 5% ws used for declring min effect or pirwise findings significnt (Verhoeven et l. 5). The list of sttisticlly significnt trnscripts ws nnotted s descried in Ruzick et l. () nd functionlly clssified ccording to Bevn et l. (998). Microrry dt ws plotted in tles s fold-chnge, indicting the difference in verge signl intensity etween groups, or plotted grphiclly s reltive signl intensity with the 76R MYC+ control tretment (wter) group set to. This rry dt (Online Resource ) hs een mde ville on the Gene Expression Omnius (GEO; gov/projects/geo/) ccession GSE37. Quntittive rel-time RT-PCR Quntittive rel-time RT-PCR ws performed including primer testing s descried in Ruzick et l. (). Online Resource 3 contins sequences of gene-specific primer pirs first reported here. qrt- PCR primers were designed t nd ll mplicon sizes were etween 8 nd nucleotides. The tomto ctin gene (BT377, LeACT) ws used s the reference control gene s it did not exhiit differentil expression mong N tretments or etween wild-type nd rmc. Reltive expression ws nlyzed ccording to the ΔΔCT method, nd stndrd error ws computed from the verge of the ΔCT vlues for ech iologicl smple. Clcultions nd sttistics The tom percentge of 5 N trcer in ech plnt prt (leves, stems, fruits nd ring roots) ws mesured y first clculting the mount of excess 5 N ove ckground. Bckground 5 N ws clculted s the verge tom% 5 N in the wter smples (men tom percent 5 N±SE in leves:.369%±.6, stems:.369% ±.5, fruits:.3688% ±.5, ring roots:.38%±.5, no significnt differences etween the two genotypes). This ckground 5 N ws then sutrcted from totl tom% 5 N. All soil nd 5 N dt ws nlyzed with three-wy, fixed fctor nlyses of vrince with genotype, nutrient ddition, nd lock s the min effects or two-wy fixed fctor nlysis of vrince without the nutrient min effect if the tretment hd not yet een pplied. All ssumptions of ANOVA were checked nd dt trnsformed s needed. Tukey-Krmer HSD test ws used to compre mens (P<.5). All dt were nlyzed using R. Results 76R MYC+ plnts exhiited AM coloniztion nd ctive symiosis Coloniztion rtes of wild-type roots (76R MYC+) were 3%, while the reduced mycorrhizl mutnt (rmc) roots were lrgely uncolonized (<%) (Fig. ) s determined y trypn lue stining nd microscopy. To estimte AM undnce or ctivity (Aono et l. ; Lmmers et l. ) using moleculr mrkers, Glomus intrrdices lkline phosphtse (ALP) nd AM fungl ß-tuulin gene expression were ssyed in the root smples using quntittive RT-PCR (qpcr). Both fungl genes were highly expressed in ll 76R MYC+ root smples ut were not detected in rmc root smples (Fig. c).

6 5 Plnt Soil () 35:5 6 The trnscriptome of ptch roots ws ssyed using Affymetrix tomto GeneChips. Across ll smples in the min experiment, 9 of the 38 proesets on the microrry were detected, indicting the expression of the corresponding genes. The rry nlysis identified 7 genes tht were differentilly regulted etween mycorrhizl wild-type nd nonmycorrhizl rmc roots irrespective of the N tretment (>.5 fold-chnge, FDR corrected P-vlue<.5) (Fig. nd Online Resource ). To test whether these expression differences were due to the AM symiosis or genetic differences etween the 76R wild-type nd rmc genotypes, we performed microrry nlysis on wild-type nd rmc root RNA smples from plnts grown in soil lcking AM spores. None of the 7 genes reported here were differentilly expressed when wild-type nd rmc trnscript profiles were compred in pot study with sterile soils in the sence of AM (dt not shown). This confirmed tht the 7 regulted genes were not due to the difference in genotype. There were 36 genes differentilly regulted etween the high NH + nd wter control tretment groups, nd 98 genes were differentilly regulted etween the high NH + nd low NH + tretment groups. Surprisingly, the rrys did not identify ny genes differentilly regulted etween the low NH + nd wter control tretments. The differentilly regulted genes were clssified into diverse functionl ctegories sed on Bevn et l. (998) (Fig. nd c, nd Online Resource 5). The effect of loclized N ptch on tomto root trnscription ws previously reported (Ruzick et l. ), nd 6 genes (3%) were similrly regulted y the N tretments in the previous greenhouse pot experiment nd this field experiment in oth wild-type nd rmc roots (Online Resource 6). The effect of AM symiosis on P nd other nutrients/ micronutrients Mycorrhizl plnts contined significntly higher totl P concentrtions in leves (Fig. 3) nd therefore my hve een less phosphte limited thn the rmc plnts. The microrry results indicte tht rmc roots were likely to e more phosphte limited s indicted y the induction of strong multigene phosphte strvtion response (PSR) (Tle ). PSR genes such s SYG/ Pho8 domin contining proteins (BI593, AJ9999, nd AJ75789), cid phosphtse TPSl (BI9966), rionuclese RNAlx (X793338), nd three phosphoric monoester hydrolses (AF35968., AJ5987., AJ5986.) were ll more highly expressed in rmc compred to mycorrhizl roots. Since the chrcterized phosphte trnsporter genes were not ll present on the tomto microrry, quntittive PCR (qpcr) ws used to test whether the mycorrhizl symiosis lso resulted in the dynmic regultion of the phosphte trnsporter (PT,, 3,, nd 5) genes (Fig. 3). PT ws not differentilly expressed etween Fig. Functionl clssifiction of tomto genes regulted y the mycorrhizl symiosis nd nitrogen tretments. Affymetrix proeset sequences were mtched to pulicly ville Gennk ccession identifictions nd ctegorized ccording to tomto or Aridopsis orthologue gene nnottions. 7 genes were differentilly regulted y the AM symiosis (). 36 genes were differentilly regulted etween the high NH + nd wter control tretments (). 98 genes were differentilly regulted etween the high NH + nd low NH + tretments (c). Blck rs represent genes more highly expressed in 76R MYC+ (), or high NH + ( nd c) roots. White rs represent genes more highly expressed in rmc (), wter control (), or low NH + (c) roots. No differentilly expressed genes were identified in the comprison etween the low NH + nd wter control tretments. Unclssified genes or with unknown function re not represented on the figure

7 Plnt Soil () 35:5 6 5 Fig. 3 Lef phosphte levels nd root expression of phosphte trnsporter genes in response to the nitrogen tretments nd mycorrhizl symiosis. Lef phosphte concentrtions were mesured in 76R MYC+ nd rmc plnts (). Expression levels of phosphte trnsporters PT (), PT (c), PT3 (d), PT (e), nd PT5 (f) were ssyed using qrt-pcr. Reltive expression ws clculted using the ΔΔCT method with ctin (LeACT) s the reference control, nd the 76R MYC+ wter control group normlized to. For PT (), different letters indicte significnt differences mong groups (-wy ANOVA, Tukey HSD). The P vlues for significnt min effectsinpt,3,,nd5 (c f) re noted (-wy ANOVA). The light nd drk grey rs represent 76R MYC+ nd rmc respectively lef % phosphte c Reltive Expression e Reltive Expression. P genotype < Phosphte Trnsporter PT 8 P genotype =. P tretment =.5 6 Phosphte Trnsporter PT.5 P genotype <. Reltive Expression d Reltive Expression f Reltive Expression Phosphte Trnsporter PT c 3 c Phosphte Trnsporter PT3 P genotype <. Phosphte Trnsporter PT5 3 P genotype <. Tle Tomto phosphte metolism nd trnsporter genes regulted y the AM symiosis etween ll 76R MYC+ nd rmc smples Phosphte trnsport nd metolism genes mycorrhizl vs. rmc Affy Proe Set ID Puttive Annottion Fold Chnge c P-vlue d Les S_t SPX domin-contining protein.77. Les.59..A_t SPX domin-contining protein.33.3 Les...S_t inorgnic phosphte trnsporter.. Les...S_t myo-inositol--phosphte synthse.5.3 LesAffx S_t phosphtse.95. Les.5..S_t rionuclese (rnlx).98. LesAffx.59..S_t SPX domin-contining protein LesAffx.878..A_t cid phosphtse TPSI Les...S_t phosphoric monester hydrolse 9.. Les.67..S_t phosphoric monester hydrolse.9. Proe Set ID; Affymetrix identifier for ech microrry proeset. Puttive Annottion; functionl nnottion sed on tomto protein function or function of Aridopsis orthologues identified with BLAST serches. c Fold Chnge; liner fold chnge (threshold >.5 fold different) in mycorrhizl roots compred to rmc roots. d P-vlue; FDR djusted P vlue threshold <.5

8 5 Plnt Soil () 35:5 6 mycorrhizl nd rmc roots in the wter control tretments, ut ws induced in rmc nd not mycorrhizl roots in the low nd high N ptches. PT expression levels were t lest three-fold higher in rmc compred to mycorrhizl roots under ll N conditions. Conversely, mycorrhizl-specific PT 3,, nd 5 were more highly expressed in mycorrhizl roots compred to rmc nd did not exhiit ny chnges due to N tretment. In ddition to totl P, the concentrtions of Cu nd S were significntly higher in mycorrhizl wild-type lef tissue (P<. nd P=.7) (Fig. nd ). Microrry nlysis detected higher expression levels of t lest one Cu trnsporter COPT (LesAffx S_t) nd Cu chperone protein (LesAffx S_t) s well s S trnsporters ST (Les.38.. S_t) nd ST (Les.379..S_t) in mycorrhizl roots (Fig. c f). Leves of the mycorrhizl wild-type plnts lso exhiited higher concentrtions of Zn nd lower concentrtions of mngnese (Mn) (dt not shown). Trnscriptome nlysis did not identify ny differentilly expressed Zn or Mn trnsporters. Additionl physiologicl nlysis of the field grown plnts including iomss, photosynthesis, nd wter reltions re presented in Online Resource 7. 76R MYC+ plnts were slightly lrger thn rmc plnts (men ± SE: 63.5±. g nd 53.8±.7 g respectively, P=.). Nutrient nlysis ws lso performed on 76R MYC+ nd rmc tomto fruit from prllel plot (see Online Resource 8 for methods nd results) from which roots were not hrvested. Tomto fruit from 76R mycorrhizl plnts hd significntly higher concentrtions of totl P, iron (Fe), nd Cu. Fruit from non-mycorrhizl rmc plnts hd higher concentrtions of Mn. AM symiosis nd hormone metolism nd signling In ddition to the effects on nutrient metolism nd uptke, the mycorrhizl symiosis resulted in the differentil expression of hormone signling nd iosynthesis genes. The methyl slicylte esterse Les.59.. S_t (6%ID Aridopsis MES Atg36) ws more highly expressed in mycorrhizl roots (Tle ). Additionl orthologous methyltrnsferses including LesAffx S_t nd Les.5..A_t were lso Fig. Shoot copper nd sulfur levels nd root expression of copper nd sulfur trnsporter genes in response to the nitrogen tretments nd mycorrhizl symiosis. Lef copper () nd sulfur () concentrtions were mesured in 76R MYC+ nd rmc plnts. Expression levels of copper trnsporter COPT (c) nd copper chperone (e), nd sulfur trnsporters ST (d) nd ST (f) were nlyzed y Affymetrix microrry. Microrry reltive signl intensities were clculted in reference to the 76R MYC+ wter tretment group nd plotted s n verge of 5 replictes. The P vlues for significnt min effects re noted (-wy ANOVA). The light nd drk grey rs represent 76R MYC+ nd rmc, respectively c e Totl Cu lef (ppm) Reltive S.I. Reltive S.I. 3 Pgenotype <. Copper Trnsporter.5 P genotype <...5. Copper Chperone Protein.5 P genotype =.5..5 Totl S lef (ppm) d f 5 Reltive S.I Reltive S.I. Sulfur Trnsporter ST P genotype =. Sulfur Trnsporter ST P genotype =.6 P genotype =. P tretment =..

9 Plnt Soil () 35: Tle Differentil regultion of tomto hormone metolism genes in root tissue y the AM symiosis Hormone metolism genes mycorrhizl vs. rmc Affy Proe Set ID Puttive Annottion Fold Chnge c P-vlue d Les.335..S_t gierellin -oxidse 9.8. Les...S_t gierellin 3-hydroxylse 5.7. Les.6..S_t gierellin -oxidse-.9. LesAffx A_t gierellin requiring Les.7..S_t coplyl diphosphte synthse.79. Les.59..S_t methylesterse.75. LesAffx S_t S-denosyl-L-methionine:croxyl methyltrnsferse.5.7 Les.5..A_t S-denosyl-L-methionine:croxyl methyltrnsferse.7.5 LesAffx.57..S_t GDSL lipse.6.6 Les.35..S_t -minocyclopropne--croxylte oxidse.. Proe Set ID; Affymetrix identifier for ech microrry proeset. Puttive Annottion; functionl nnottion sed on tomto protein function or function of Aridopsis orthologues identified with BLAST serches. c Fold Chnge; liner fold chnge (threshold >.5 fold different) in mycorrhizl roots compred to rmc roots. d P-vlue; FDR djusted P vlue threshold <.5 more highly expressed in mycorrhizl roots. A slicylic cid-repressed GDSL-lipse (LesAffx.57..S_t) ws more highly expressed in rmc roots. Genes involved in gierellin (GA) synthesis, signling, nd degrdtion were induced in 76R MYC+ roots (Tle ). These included GA -β dioxygense, GA -oxidse-, GA 3-β hydroxylse, GA-requiring 3, nd GArequiring coplyl diphosphte synthse (Les S_t, Les.6..S_t, Les...S_t, LesAffx A_t, nd Les.7..S_t respectively). Ptch root smples were nlyzed for chnges in hormone concentrtions with liquid chromtogrphy- tndem mss spectrometry (LC/MS/MS) to test whether trnscriptionl chnges in hormone iosynthesis nd secondry metolism genes correlted with phytohormone concentrtions. With these methods we were le to nlyze indole cetic cid (IAA), scisic cid (ABA), jsmonic cid (JA), jsmonic cid isoleucine (JA-Ile), nd slicylic cid (SA) (Fig. 5). IAA nd ABA concentrtions were not significntly different mong N tretment nd mycorrhizl coloniztion sttus (Fig. 5 nd ). SA concentrtions were ~.5-fold higher in mycorrhizl roots compred to rmc (P<., Fig. 5e). The N tretments lso ffected root hormone levels including higher levels of JA nd JA-Ile in the low N tretment (Fig. 5c nd d) which ws independent of the coloniztion sttus of the roots. Mycorrhizl roots exhiited slightly higher levels of JA-Ile compred to rmc roots specificlly in the low N tretment (P=.7, Fig. 5d). Soil N trnsformtions nd root uptke Twenty-four hours fter 5 NH + -N injection into the rings, soil NH + concentrtions were nerly 6- nd 3.35-fold higher in the high NH + + nd low NH tretment ptches compred to the wter control, respectively (tretment P<., Fig. 6). However, soil NH + concentrtions were pproximtely 3% nd 6% of the clculted pplied mounts of the high NH + nd the low NH + tretments, respectively. This indictes tht rpid chnges in plnt-microe-soil NH + trnsformtions hd occurred during this time - period. Soil NO 3 levels were 5-fold nd.75-fold higher in the high NH + nd low NH + tretment ptches compred to the wter control indicting sustntil nitrifiction in h (Fig. 6). Tomto roots took up significnt mounts of leled 5 N from the soil ptches nd trnslocted this N to shoot tissue over time. Ptch roots from the low nd high NH + tretment groups contined higher tom% 5 N compred to the wter controls t the -hr hrvest (Fig. 6c). The rpid increse in root N uptke corresponded with the lrge effect on root gene expression (Fig. nd c). Less thn 7% of the dded 5 N ws recovered in the shoots of either genotype fter h (dt not shown). Interestingly, rmc showed trendtowrdshighertom% 5 N in the ring roots compred to 76R MYC+ (P=.8) in oth the low nd high N tretments (Fig. 6c). When uptke of 5 N ws followed over the course of week in prllel

10 5 Plnt Soil () 35:5 6 Fig. 5 Root plnt hormone concentrtions in response to the nitrogen ptch nd mycorrhizl symiosis. Concentrtions of plnt hormones indole cetic cid (IAA) (), scisic cid (ABA) (), jsmonic cid (JA) (c), jsmonic cid isoleucine (JA-Ile) (d), nd slicylic cid (SA) (e) were mesured y LC/MS/MS in 76R MYC+ nd rmc roots from the vrious N tretment plots, nd clculted y dividing y the fresh weight of the root smple. The P vlues for significnt min effects in JA (c) nd SA(e) re noted (-wy ANOVA). For JA-Ile (d), different letters indicte significnt differences mong groups (-wy ANOVA, Tukey HSD). The light nd drk grey rs represent 76R MYC+ nd rmc, respectively. n.s.d; no significnt difference. g f.w; grms fresh weight ng IAA /g f.w. c ng JA / g f.w. e ng SA / g f.w n.s.d P tretment =.5 P genotype <. P tretment =.35 ng ABA / g f.w. d ng JA-Ile / g f.w.. n.s.d set of high NH + tretment nd wter control plnts, the tom% 5 N in the leves incresed significntly over time in the high NH + tretment plnts, ut no differences were found etween mycorrhizl nd rmc lef tissue (time P=.6, Fig. 6d). Generl trnscriptionl responses to the N ptch Despite the trend of higher short-term 5 Nuptkefrom the nitrogen ptch y rmc roots, mycorrhizl plnts hd higher totl N concentrtions in shoot tissue (Fig. 7). A serch of the tomto genome sequence ( nomics.net/out/tomto_project/) identified novel tomto mmonium trnsporter gene (AC538_5.., LeAMT) on chromosome 9 tht ws 68% identicl to mycorrhizl root specific L. jponicus LjAMT. (ABO83). Expression nlysis with qpcr showed tht LeAMT ws exclusively expressed in mycorrhizl roots, lthough ws not significntly regulted y the NH + tretments (Fig. 7). A second tomto mmonium trnsporter (AC_., LeAMT5) tht shres 6.% mino cid identity with LeAMT ws lso exclusively expressed in mycorrhizl roots nd not significntly regulted y the NH + tretments (Fig. 7c). Phylogenetic nlysis of the tomto AMT gene fmily confirmed tht LeAMT nd LeAMT5 oth fit into n rusculr mycorrhizl-specific clde tht includes mycorrhizl root specific AMTs L. jponicus LjAMT. (ABO83) nd M. trunctul MtAMT (ABO83) (Online Resource 9). We serched for ptterns in the microrry nd qrt-pcr dt tht might indicte differentil response to the NH + tretments depending on the mycorrhizl coloniztion of the roots (-wy ANOVA, see mterils nd methods). The mjority of genes regulted y the NH + tretments were not ffected y the symiosis (Online Resource 5). However, numer of N metolism genes were found to e differentilly regulted etween mycorrhizl nd rmc roots specificlly under the most limiting N conditions (wter control tretment) (Fig. 7d k), nd these expression differences etween mycorrhizl nd rmc roots were less pronounced or sent when N(6.5or65μg NH + -N g soil) ws dded to the ptch in the NH + tretment groups. Glutmine dehydrogense GDH (Les.69..S_t) (Fig. 7d), cytosolic glutmine synthetse GS (Les... A_t) (Fig. 7e) nd sprgine synthetse AS

11 Plnt Soil () 35: µg NH + -N /g ring soil µg NO 3 - -N /g ring soil c tom % 5 N ring roots c c Fig. 6 Soil nd root N dynmics nd uptke in response to the nitrogen tretments nd mycorrhizl symiosis. Ring soil ws nlyzed for NH + () nd NO 3 () concentrtions. Plnts were nlyzed for percent recovery of 5 N in the roots (c). Atom% recovery 5 N differed in roots y tretment (P<.) nd showed trend towrd higher tom% 5 Ninrmc compred to 76R MYC+ (P=.8). 5 N uptke ws lso mesured in individul leflets over time on prllel set of plnts nd displyed s the tom% excess of 5 N in the tretment group ove the wter control nturl undnce (n..) 5 Nlevel (d). Letters indicte significnt differences mong tretments (P<.5).Lightnddrkgreyrsrefor76RMYC+nd rmc, respectively induced y the N tretments, ut were specificlly more highly expressed in rmc roots compred to 76R MYC+ roots in the wter control tretment. Furthermore, while the expression levels of nitrte trnsporters NRT.-like (TC7669) (Fig. 7i) nd NRT3. (LesAffx.696..S_t) (Fig. 7j) did not respond to the N tretments, oth genes were expressed more highly in rmc roots compred to 76R MYC+ specificlly in the wter control tretment. The NH + trnsporter AMT (Les.36..S_t) (Fig. 7k) ws induced y the N tretments in mycorrhizl roots, ut not in rmc roots. Under the lower N conditions of the wter control AMT ws more highly expressed in rmc roots s compred to the wild-type roots. These expression differences etween 76R MYC+ nd rmc roots under the wter control conditions re summrized nd put into the context of N uptke, ssimiltion nd metolism in Fig. 8. d tom % 5 N in leves > n h h 8h h (Les.37..S_t) (Fig. 7f) were induced in oth 76R MYC+ nd rmc roots y the N tretments, ut under the wter control conditions, ll three were more highly expressed in 76R MYC+ roots compred to rmc roots. Nitrte reductse NR (Fig. 7g) nd nitrite reductse Nii (LesAffx.535..S_t) (Fig. 7h) were lso Discussion Trnscriptome nd metolome chnges in response to the AM symiosis The complex reltionship etween plnt roots nd mycorrhizl fungi necessittes the continuous exchnge of signls nd the regultion of plnt genes to control downstrem chnges in plnt development nd physiology. The higher concentrtion of P in mycorrhizl plnts oserved in this study is consistent with previous field oservtions (Cvgnro et l. 8). In this study the non-mycorrhizl mutnt (rmc) exhiited more pronounced phosphte strvtion response which included the incresed expression of root P trnsporters PT nd PT. These findings re in greement with the well ccepted model of the suppression of the generl phosphte strvtion

12 56 Plnt Soil () 35:5 6 % N in shoots Totl shoot % N P genotype =.8 Reltive Expression 3 Ammonium Trnsporter AMT P genotype <. c Reltive Expression 5 3 Ammonium Trnsporter 5 P genotype <. d Glutmine synthetse GS e Asprgine Synthetse f 6.5 Reltive Expression 8 **.5 Reltive S.I...5. *** *.5. Reltive S.I Glutmte dehydrogense ** g Reltive S.I. 3 ** Nitrte Reductse NR h Reltive S.I ** Nitrite Reductse Nii i Reltive Expression 3 Nitrte Trnsporter.-like P genotype =.9 j Reltive S.I * Nitrte trnsporter 3. k Reltive Expression 5 3 Ammonium trnsporter ** Fig. 7 Shoot N levels nd root expression of N trnsporter, ssimiltion nd metolism genes in response to the nitrogen tretments nd mycorrhizl symiosis. Lef N concentrtion ws mesured in 76R MYC+ nd rmc plnts (). qpcr nd/or + microrrys were used to ssy the expression level of NH trnsporter LeAMT (), NH + trnsporter LeAMT5 (c), glutmine synthetse (GS) (d), sprgine synthetse (ASN) (e), glutmte dehydrogense (GDH) (f), nitrte trnsporter NRT.-like (g), nitrte trnsporter 3. (h), nitrte reductse (i), nitrite reductse (j), nd mmonium trnsporter AMT (k). response nd certin root Pi trnsporters mycorrhizl roots (Bucher 7). qpcr reltive expression ws clculted using the ΔΔCT method with ctin (LeACT) s the reference control, nd the 76R MYC+ wter tretment group normlized to. Microrry reltive signl intensities were clculted in reference to the 76R MYC+ wter tretment group nd plotted s n verge of 5 replictes. The P vlues for significnt min effects re noted nd sterisks represent significnt difference etween 76R MYC+ nd rmc t the given pirwise comprison (***<., **<., *<.5) (-wy ANOVA, tukey HSD). Light nd drk grey rs re for 76R MYC+ nd rmc, respectively Previous studies hve lso shown tht the mycorrhizl symiosis increses oth plnt mcro- nd

13 Plnt Soil () 35: Fig. 8 Model of nitrogen uptke nd ssimiltion gene regultion in response to the mycorrhizl symiosis under lower soil N conditions. Mycorrhizl roots exhiit drmticlly ltered N uptke, ssimiltion nd mino cid metolism trnscriptionl profile compred to non-mycorrhizl roots in the control tretment, suggesting incresed mycorrhizl-medited N uptke nd ssimiltion nd decresed direct root-medited N uptke nd ssimiltion. Yellow: rusculr mycorrhizl fungl extrrdicl mycelium nd intrrdicl mycelium comprtments. Grey: Plnt root cell. Green: plstid. Gene colors indicte micronutrient concentrtions (Cvgnro et l. 8; Liu et l. ). When soil S vilility is low, the AM symiosis cn increse S levels in plnt tissues (Bnerjee et l. 3; Guo et l. 6). Recent work demonstrted the role of AM in incresing root S content through the trnscriptionl regultion of puttive fungl sulfte permese (Allen nd Shchr- Hill 9). Studies on mycorrhizl regultion of copper nd mmonium trnsporters in Medicgo trunctul, nd NH +, S, nd K trnsporters in Lotus jponicus identified puttive trnsporters ut did not ssy corresponding plnt tissue nutrient levels (Gomez et l. 9; Guether et l. 9). This study nd previous work with these tomto genotypes grown on the sme orgnic frm soil showed higher concentrtions of Cu nd S in shoot tissue (Cvgnro et l. 8) of the mycorrhizl roots compred to nonmycorrhizl mutnt rmc. This work provides link expression level differences etween 76R MYC+ nd rmc smples in the wter control tretment conditions (lue with white text = higher in rmc, ornge with lck text = higher in 76R MYC+). Thicker rrows suggest shift towrd specified metolic pthwy sed on the trnscriptionl dt. Arevitions: GS glutmine synthetse, GOGAT glutmte synthse, ASN sprgine synthetse, AMT mmonium trnsporter, NRT nitrte trnsporter, NR nitrte reductse, NRT nitrite reductse, GDH glutmte dehydrogense, AspAT sprtte minotrnsferse etween the mycorrhizl-induced plnt S nd Cu trnsporters nd the incresed S nd Cu concentrtions in lef tissue of the mycorrhizl wild-type plnts. Further work will e necessry to estlish cusl reltionship etween these cndidte mycorrhizl-induced trnsporters nd incresed shootnutrientlevels. Plnt hormones nd the AM symiosis Hormones regulte mny plnt responses nd re thus strong cndidtes for regultion of physiologicl processes during coloniztion. AM fungl coloniztion hs een shown to induce numerous hormone iosynthesis nd signling genes nd lter hormone levels (Grcí Grrido et l. ; Huse et l. 7; López-Ráez et l. ; Shul-Keinn et l. ). In this study multiple genes were induced in mycorrhizl

14 58 Plnt Soil () 35:5 6 roots involved in GA metolism (gierellin - oxidse, gierellin -oxidse, gierellin 3-oxidse, GA-requiring 3 cytochrome P5 ent-kurene oxidse, nd GA-requiring coplyl P synthse). Studies in M. trunctul nd tocco hve lso shown tht the GA iosynthesis pthwy is induced y AM symiosis (Hohnjec et l. 5; Shul-Keinn et l. ), suggesting conserved role for GA in the AM symiosis cross plnt species nd ecologicl settings. GA my ply role in repressing P strvtion response (Jing et l. 7), which is lso repressed in the wild-type mycorrhizl tomto roots (Tle ). Higher concentrtions of GA in mycorrhizl roots my increse the strength of the crohydrte sink tht is creted nd utilized y the fungus (Blee nd Anderson 998). The simultneous up-regultion of oth GA iosynthesis nd degrdtion enzymes in the present study lso suggests dynmic role for this plnt hormone in the AM symiosis tht my vry y root cell type or throughout the course of ruscule development. Mycorrhizl coloniztion lso impcts plnt hormones involved in defense nd stress responses such s SA (Gutjhr et l. 8; Huse et l. 7). Mycorrhizl plnts hd significntly higher concentrtions of SA in their roots nd significntly higher expression of multiple methyltrnsferses tht my function in SA metolism (Vlot et l. 9); however genes in the SA iosynthesis pthwy were not differentilly expressed. Previous studies lso support the ide tht SA levels increse in roots t erly stges of AM interction when AM my trigger defense response in the root (Blilou et l. 999; ). Considering tht of the 7 genes differentilly regulted etween mycorrhizl nd rmc roots function in disese/defense response (Fig. ), it is possile tht the AM symiosis induced n SAmedited defense response in mycorrhizl roots (Liu et l. 3). Nitrogen trnsformtions nd metolism Fertilizer ppliction on conventionl nd orgnic frms often occurs in pulses nd ptches similr to the nutrient dditions pplied in this experiment. Rpid N uptke occurs with ckdrop of highly complex soil processes; the roots must rpidly sense N nd compete successfully with soil microil nd pssive soil processes such s soil microil N ssimiltion nd nitrifiction, leching nd denitrifiction (Burger nd Jckson 5; Run nd Johnson 999). N trnsformtion in the soil ws rpid with much of the dded NH + eing trnsformed to NO 3 within h. Becuse soil moisture ws < 5% wter filled pore spce, the loss of NO 3 vi denitrifiction in this experiment ws ssumed to e negligile (Linn nd Dorn 98). Over h, plnts were le to rpidly cpitlize on the nutrient ptch y tking up pproximtely seven percent of the pplied 5 N. The high competition etween roots nd soil microes, the reltively low root length densities in the rings, nd the short h tretment time my ll contriute to the lower recovery rtes of 5 N compred to other studies (Burger nd Jckson 5). In n erlier greenhouse study (Ruzick et l. ), we reported on the trnscriptionl response to n enriched N ptch fter 53 h of mycorrhizl roots of tomto plnts grown in pots under greenhouse conditions. Tht response included more thn 55 genes, nd suggested n ctive nd sustined response to oth NH + nd NO 3 in the ptch fter 53 h. In this present study, we identified sixty one genes s eing similrly regulted etween the current field study nd the erlier greenhouse pot study (Online Resource 6), highlighting the vlue of field experiments to vlidte trnscriptionl responses mesured under controlled environment conditions. Mny of the overlpping genes encode proteins tht function in nitrogen metolism, protein synthesis nd turnover, nd trnscriptionl regultion. These genes my represent core trnscriptionl response to N uptke nd ptch utiliztion cross vrile environmentl or ecologicl conditions. Future work could utilize these genes s mrkers to identify roots tht re responding to soil N vilility, even when soil microil processes of nitrifiction nd immoiliztion re rpidly chnging. AM-medited N uptke nd metolism Puttive mmonium trnsporters LeAMT nd LeAMT5 were expressed exclusively in wild-type mycorrhizl roots nd my e the key plnt mycorrhizl NH + trnsporters in tomto. Orthologous Lotus nd Medicgo AMT genes re lso induced y the symiosis, nd LjAMT. ws shown to function s on fide mmonium trnsporter (Gomez et l. 9; Guether et l. 9). In contrst to other non-

15 Plnt Soil () 35: symiosis specific tomto mmonium trnsporters tht re regulted y the presence or sence of NH + nd NO 3 (Ruzick et l. ; Von Wirén et l. ), mycorrhizl-specific LeAMT nd LeAMT5 were similrly expressed under ll N tretment conditions. This suggests tht the mycorrhizl symiosis my supply roots with dditionl N in constitutive mnner regrdless of soil N sttus in contrst to the mycorrhizl Pi uptke mechnism tht is repressed y plnt nd soil Pi (Ngy et l. 9). It is lso possiility tht these mycorrhizl-specific N trnsporters could e regulted y plnt N sttus, which ws similr in ll of the tretments in this study presumly due to the short h exposure to the mmonium dded to the soil ptch. The trnscriptionl regultion of plnt N uptke, ssimiltion, nd mino cid metolism genes in this study contriute new informtion to the plnt response to AM symiosis N uptke (Fig. 8). Previous models of the AM symiosis N uptke mechnism hve detiled the role of the rusculr mycorrhizl fungus prtner (Govindrjulu et l. 5; Jin et l. 5; Tinetl. ). The higher totl N concentrtion of mycorrhizl wild-type shoot tissue (Fig. 7) suggested tht mycorrhizl plnts were tking up nd ssimilting more N thn the non-mycorrhizl plnts throughout the growing seson. Bsed on tomto root trnscriptome nlysis, this increse ppers to e fcilitted through the mycorrhizl-specific AMT nd AMT5 mmonium trnsporters, lthough due to the prtil genome coverge of the tomto GeneChip it is possile tht other trnsporters my ply role. Consequently, cytosolic NH + supplied vi these mycorrhizl-specific trnsporters could ct s signling molecule in the root cell under reltively low externl N conditions, triggering the induction of N ssimiltion nd mino cid metolism enzymes glutmte dehydrogense, cytosolic glutmine synthetse, nd sprgine synthetse (Fig. 8, ornge oxes). This grees with the previous oservtion of incresed glutmine synthetse expression in crrot upon tretment of fungl extrrdicl mycelium with KNO 3 (Tin et l. ). Perhps more intriguing re the effects of the mycorrhizl symiosis on non-symiosis specific NO 3 nd NH + trnsporters NRT3., NRT.-like, nd AMT, s well s NO 3 reductse nd NO reductse (Fig. 8, lue oxes). These genes were ll repressed in mycorrhizl roots in the wter control, suggesting the down-regultion of the direct root NO 3 nd NH + uptke pthwys in the presence of n ctive supply of NH + derived from mycorrhizl fungi. Cumultively, these expression ptterns fit into model where dditionl NH + supplied vi the mycorrhizlmedited uptke pthwy (i.e. LeAMT nd LeAMT5) induces N ssimiltion gene expression to utilize the dditionl cytosolic NH + nd reduces the need for other direct root-medited inorgnic N uptke pthwys. The pprent shift wy from direct rootmedited NO + 3 uptke to mycorrhizl-medited NH uptke my represent n energeticlly dvntgeous venue for N uptke (Bloom et l. ). Alterntively, it my lnce the different uptke pthwys nd increse the NH + :NO 3 rtio, which my result in n enhnced growth response (Lopes nd Arus 8). Given the more moile nture of NO 3, it is interesting to speculte tht non-mycorrhizl roots depend more on encounters with moile NO 3 while mycorrhizl roots utilize fungl N uptke from orgnic N, NH + or NO 3 (Hodge nd Fitter ; Tinetl.). This study quntified chnges in roots fter short pulse of nitrogen. However, it would e interesting to test how mycorrhizl N uptke pthwys nd mycorrhizl coloniztion re ffected when plnts re grown for long periods under high N. Previous studies hve demonstrted tht sufficient Pi reduces oth AM coloniztion nd the positive effects of the symiosis on plnt growth (Bruce et l. 99; Mendoz nd Pgni 997), ut studies under high or sufficient N vilility report oth positive nd negtive effects on AM (Jckson et l. ; Olsson et l. 5; Tkcs et l. 7). Implictions for sustinle griculture Mycorrhize clerly ply key role in plnt nutrition, prticulrly N nd Pi, nd yet they re lrgely sent from conventionl frms ecuse of the use of fertilizers nd pesticides. Orgnic frms tend to hve much higher undnces of AM fungi nd my e etter poised to enefit from their presence, s mycorrhize cn improve soil ggregtion (Rillig nd Mummey 6), decrese leching (Asghri et l. 5), increse the efficiency of nutrient cycling (Conyers nd Moody 9), nd improve the nutritionl qulity of crops under certin conditions (Cvgnro et l. 8). Our study of the AM root trnscriptome identified key N uptke nd metolism processes tht were influenced y the AM symiosis

16 6 Plnt Soil () 35:5 6 in lower N soil conditions, highlighting the importnce of n ctive AM symiosis to plnt nutrient uptke in lower input griculture. More rodly, our results suggest tht future field AM-root trnscriptome studies my e useful to identify mechnisms y which mycorrhize improve the efficiency of nutrient cycling, nd my ultimtely guide frmers to reduce their inputs nd reduce nutrient losses to the environment (Fitter et l. ). As griculture moves towrd ecologicl intensifiction prctices tht include crop rottions, cover crops, nd reduced pesticide, chemicl fertilizer, nd wter inputs, mycorrhize will lmost certinly ply lrger nd more importnt role on the frmscpe nd in sustinle griculture. Acknowledgements This reserch ws supported y the Ntionl Science Foundtion Environmentl Genomics grnt numer to DPS nd LEJ. We thnk Julie Chou, Kimerly Jcos, Joel Krmer, Julien Linres, Wlter Lopez, Annie Young-Mtthews nd other memers of the Jckson l for their ssistnce with field work nd smple processing, the University of Missouri DNA Core Fcility for microrry processing, the Dnforth Center Proteomics nd Mss Spectrometry Fcility for plnt hormone nlysis, nd Luren McIntyre for consulttion regrding microrry nlysis. References Allen JW, Shchr-Hill Y (9) Sulfur trnsfer through n rusculr mycorrhiz. Plnt Physiol 9:59 56 Aono T, Mldondo-Mendoz IE, Dewre GR, Hrrison MJ, Sito M () Expression of lkline phosphtse genes in rusculr mycorrhizs. New Phytol 6:55 53 Asghri HR, Chittleorough DJ, Smith FA, Smith SE (5) Influence of rusculr mycorrhizl (AM) symiosis on phosphorus leching through soil cores. Plnt nd Soil 75:8 93 Bnerjee R, Evnde R, Kil Ã, Ojh S, Tok S (3) Rection mechnism nd regultion of cystthionine βsynthse. 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