Jasmonate-dependent depletion of soluble sugars compromises plant resistance to Manduca sexta

Transcription

1 Reserh Jsmonte-dependent depletion of solule sugrs ompromises plnt resistne to Mndu sext Rirdo A. R. Mhdo,, Crl C. M. Are,,3, Aigil P. Ferrieri,, In T. Bldwin nd Mtthis Er,4 Root Herivore Intertio Group, Mx Plnk Ititute for Chemil Eology, H-Kn oll-str. 8, 7745 Jen, Germny; Deprtment of Moleulr Eology, Mx Plnk Ititute for Chemil Eology, H-Kn oll-str. 8, 7745 Jen, Germny; 3 Deprtment of Entomology, Universidde Federl de Vißos, Avenid Peter Henry Rolfs, Vißos, Brzil; 4 Ititute of Plnt Sienes, University of Bern, Altenergrin, CH-33 Bern, Switzerlnd Author for orrespondene: Mtthis Er Tel: Emil: mtthis.er@ips.unie.h Reeived: 4 Deemer 4 Aepted: 8 Jnury 5 New Phytologist (5) 7: 9 5 doi:./nph.3337 Key words: rohydrtes, iet nutrition, jsmontes, Mndu sext, Niotin ttenut, primry metolism. Summry Jsmontes regulte plnt seondry metolism nd herivore resistne. How they influene primry metolites nd how this my ffet herivore growth nd performne re not well understood. We profiled sugrs nd strh of jsmonte iosynthesis-defiient nd jsmonte-ieitive Niotin ttenut plnts nd mnipulted lef rohydrtes through geneti engineering nd in vitro omplementtion to ssess how jsmonte-dependent sugr umultion ffets the growth of Mndu sext terpillrs. We found tht jsmontes redue the otitutive nd herivore-indued onentrtion of gluose nd frutose in the leves ross different developmentl stges. Diurnl, jsmontedependent inhiition of invertse tivity ws identified s likely mehnism for this phenomenon. Contrry to our expettion, oth in plnt nd in vitro pprohes showed tht the lower sugr onentrtio led to inresed M. sext growth. As oequene, jsmonte-dependent depletion of sugrs rendered N. ttenut plnts more suseptile to M. sext ttk. In onlusion, jsmontes re importnt regultors of lef rohydrte umultion nd this determines herivore growth. Jsmonte-dependent resistne is redued rther thn enhned through the suppression of gluose nd frutose onentrtio, whih my ontriute to the evolution of divergent resistne strtegies of plnts in nture. Introdution Jsmontes regulte plnt respoes to ioti nd ioti stress nd influene plnt growth nd development. They re prt of the regultory networks of plnt symiont (Pozo & Azon-Aguilr, 7; Stein et l., 8; Jos et l., ), plnt pthogen (Lndgrf et l., ) nd plnt herivore intertio (reviewed y Wu & Bldwin, ), nd re involved in the regultion of seed germintion (Corineu et l., 988), root growth nd development (Stswik et l., 99), lef movement (Nkmur et l., 6) nd flower development (Li et l., 4). Perhps the est known funtion of jsmontes is their stimultory effet on plnt seondry hemistry. Plnts impired in jsmonte prodution or pereption generlly disply redued levels of otitutive nd indued seondry metolites (Chen et l., 6; Pshold et l., 7; Shoji et l., 8; Zhng et l., ). Although our understnding of severl spets of jsmonte signling is inresing, knowledge out its possile role s regultor of primry metolism in plnts is unler. Reently, lef gluose nd frutose onentrtio were found to e otitutively higher nd less depleted in respoe to simulted Mndu Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust sext herivory in jsmonte iosynthesis-defiient Niotin ttenut plnts, n effet tht n e mimiked y the exogenous pplition of jsmoni id (JA; Mhdo et l., 3). Moreover, exogenous jsmonte pplition to the leves redued lef strh onentrtion in poplr trees, stem sugrs in tulip, lef sugrs in too, nd lef sugrs nd mino ids in ge (Bst et l., 5; Skrzypek et l., 5; vn Dm & Oomen, 8; Hnik et l., ; Tytgt et l., 3), suggesting tht jsmontes might t s negtive regultors of plnt primry metolism. By ontrst, strh onentrtio in jsmonte signlingimpired too plnts were signifintly lower (Wng et l., 4) nd jsmonte pplition to the leves indued mino ids in too leves (Hnik et l., ), suggesting tht jsmontes n lso promote strh nd mino id umultion. A detiled nlysis of primry metolites in jsmonte signlingimpired plnts is therefore required to lrify the potentil role of endogenous jsmontes in the regultion of plnt primry metolism. Cotitutive nd indued jsmonte levels hnge over plnt development (Adl et l., ; Diezel et l., ), phenomenon tht orreltes with redution in the mgnitude of indution of jsmonte-dependent seondry metolites nd defeive protei (vn Dm et l., ; Kur et l., ; New Phytologist (5) 7: 9 5 9

2 9 Reserh New Phytologist Onkokesung et l., ). It is therefore possile tht the impt of jsmontes on lef rohydrtes is dependent on plnt s developmentl stge. Sugrs re the dominnt solule lef rohydrtes of plnts. They re produed through the inorportion of ron dioxide (CO ) into riulose-,5-isphosphte (RuBP) y the tion of riulose-,5-isphosphte roxylse/oxygee (RuBisCO), followed y the spontneous formtion of two moleules of 3- phosphoglyeri id (3PGA). RuBisCO tivse (RCA) tivtes RuBisCO y removing inhiitory sugr phosphtes from the tive site. 3PGA is susequently onverted to gluose, frutose, surose nd strh vi severl enzymti steps. Surose nd strh n e stored, trported nd/or metolized further (Brun et l., 4). Solule sugrs nd strh umulte during the dy nd re tolized during the night to meet the energy demnd of the plnt. Therefore, their onentrtio rise nd fll in diurnl mnner. Diurnl ptter therefore need to e tken into ount when studying the impt of jsmontes on lef rohydrtes. Phytophgous iets feed on plnts to quire nutrients to fuel growth, development nd reprodution, nd re therefore ffeted diretly y the metoli mke up of their food soure. Both primry nd seondry metolites influene iet performne (Roeder & Behmer, 4). Seondry metolites re diretly toxi or redue the digestiility of the plnt mteril in quntittive mnner (Bennett & Wllsgrove, 994). The influene of primry metolites on herivores is more ontext dependent (Behmer, 8). The rtio etween rohydrtes nd protein, for itne, determines iet growth in nonliner fshion, with suoptiml rtios leding to rpid redution in growth rtes (Thompson & Redk, ; Simpson & Ruenheimer, 9; Roeder & Behmer, 4). Furthermore, protein nd rohydrte rtios influene the toxiity of plnt seondry metolites (Ruenheimer & Simpson, 99; Ruenheimer, 99; Simpson & Ruenheimer, ). Most studies on iet nutrition hve een rried out in hemilly defined rtifiil environments. However, plnts s food soures in nture re inherently vrile. Herivore ttk, for itne, lters nitrogen nd ron dynmis (Arnold & Shultz, ; Bst et l., 5; Gomez et l., ; Appel et l., ), whih often results in drmti hnges in primry nd seondry metolite pools (Bst et l., 5; Skrzypek et l., 5; Shwhtje et l., 6; Steinrenner et l., ; Gomez et l., ; Mhdo et l., 3) tht might ffet the nutritionl qulity of folir tissue nd ould potentilly ffet herivore nutrition. If we re to understnd the importne of rohydrtes for iet nutrition, omining in vitro ssys with experiments in plnt would therefore e promising pproh. One pproh to mnipulte plnt hemistry is to trget defeive signls. Jsmontes, for itne, hve een silened in numer of plnt speies, nd the suseptiility of the jsmonte signling-impired plnts to herivores hs susequently een ttriuted to defiienies in seondry metolite prodution nd umultion (Steppuhn et l., 4; Pshold et l., 7; Steppuhn & Bldwin, 7; Heiling et l., ). Given tht jsmontes lso regulte primry metolites in plnts (Mhdo et l., New Phytologist (5) 7: 9 5 3; Wng et l., 4) nd tht the primry metolites n e eqully importnt for iet performne (Fertrom, 987; Cohen et l., 988; Wlduer & Friedmn, 99; Thompson & Redk, ; Simpson & Ruenheimer, 9; Roeder & Behmer, 4), the question rises s to whether they ould e respoile for the oserved suseptiility of jsmonte-defiient plnts. We investigted this potentilly overlooked spet of plnt herivore intertio y studying the role of jsmontes in the regultion of rohydrte umultion in N. ttenut leves, inluding potentil underlying mehnisms, nd the ontriution of jsmonte-dependent rohydrte depletion to herivore resistne. To wer the first question, we mesured sugr onentrtio nd invertse tivity in N. ttenut genotypes tht re impired to different degrees in their jsmonte iosynthesis, signling nd/or pereption. To wer the seond question, we evluted M. sext growth when feeding on plnts, rtifiil nd semi-rtifiil diets with different sugr onentrtio. Our results revel tht solule sugr onentrtio redue rther thn enhne jsmonte-dependent plnt resistne. Mterils nd Methods Plnt mteril Trgeni inverted repet (ir) nd empty vetor () ontrol (A-3-9-) Niotin ttenut Torr. Ex. Wtson plnts were used in this study. The hrteristis of these previously hrterized different genotypes re summrized in Tle. In ddition, we produed hemizygous ross etween inverted repet llene oxide ylse (; line A ) nd inverted repet riulose-,5-isphosphte roxylse/oxygee tivse (irrca; line A ) lines y removing nthers from flowers of irrca plnts efore pollen mturtion nd pollinting the stigms with pollen from plnts. Plnting onditio Before plnting, ll seeds were surfe sterilized nd germinted on Gmorg s B5 medium (Kr ugel et l., ). Ten-dy old seedlings were trferred to Teku pots for nother d (P oppelmnn GmH & Co. KG, Lohne, Germny) efore plnting them into -l pots filled with wshed snd or stndrd sustrte. Plnts were grown t 45 55% reltive humidity nd 4 6 C during dys nd 3 5 C during nights under 6 h of light (6: : h). Plnts were wtered twie every dy. Solule sugr, strh nd protein onentrtio in jsmonte signling-impired lines ross different developmentl stges To investigte the possile role of jsmontes in the regultion of primry metolism in N. ttenut, we mesured gluose, frutose, surose, strh nd solule protein onentrtio in the rosette leves of jsmonte iosynthesis-defiient nd jsmonte pereption-impired inverted repet orontine ieitive (ircoi) plnts. As endogenous jsmonte levels hnge Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust

3 New Phytologist Reserh 93 Tle Chrteristis of the inverted repet (ir) Niotin ttenut trgeni lines used in the present study Genotype Gene silened Impired funtion Phenotype Referene irsipk Sliyli id-indued protein kie Erly jsmonte signling Redued levels of jsmontes Meldu et l. (9) irwipk Wound-indued protein kie irgla Glyerolipse A Jsmonte iosynthesis Bonventure et l. () iraos Allene oxide synthse Kllenh et l. () Allene oxide ylse iropr3 -oxo-phytodienoi id redutse irjar4/6 JA-Ile synthetse Redued levels of JA-Ile Wng et l. (8) ircoi Corontine-ieitive JA-Ile pereption Redued JA-Ile pereption Pshold et l. (7) irrca Riulose-,5-isphosphte Photosynthesis Redued photosyntheti tivity Mitr & Bldwin (8) roxylse/oxygee tivse 9 irrca Allene oxide ylse nd riulose-,5-isphosphte roxylse/oxygee tivse Jsmonte iosynthesis nd sugr metolism Redued sugr onentrtio ompred with plnts Present study JA-Ile, jsmonoyl-l-isoleuine. over plnt development (Adl et l., ), we mesured sugr onentrtio t four different developmentl stges: erly rosette (3 d fter germintion; DAG), rosette (38 DAG), elongtion (44 DAG) nd erly flowering (5 DAG). Sugr nd strh onentrtio were quntified s desried y Mhdo et l. (3). Briefly, solule sugrs were extrted from plnt tissue using 8% (v/v) ethnol, followed y n inution step ( min t 8 C). Pellets were re-extrted twie with 5% (v/v) ethnol ( min t 8 C). Superntnts from ll extrtion steps were pooled together, nd surose, gluose nd frutose were quntified enzymtilly s desried y Velterop & Vos (). The remining pellets were used for n enzymti determintion of strh (Smith & Zeemn, 6). In ddition, totl solule protein ws quntified (Brdford, 976). As protein soluility is ffeted y ph, totl solule protein levels my e underestimted y this method. Five independent replites of eh genotype nd developmentl stge were nlyzed. Plnt leves were hrvested t 3: h nd flsh frozen in liquid nitrogen for nlysis. Cotitutive jsmonte nd solule sugr onentrtio in jsmonte-defiient plnts To ssess the importne of jsmontes for sugr umultion, we evluted eight different genetilly engineered lines tht differ in their pity to produe jsmontes euse they re defiient in either jsmonte iosynthesis or in the upstrem signling network. We mesured gluose, frutose nd surose onentrtio, s well s otitutive JA nd jsmonoyl-l-isoleuine (JA-Ile), in the leves of rosette stge plnts of ll genotypes. Phytohormone mesurements were rried out s desried y Mhdo et l. (3). Plnts were hrvested t : h (n = 5). Sugrs were then orrelted with phytohormone levels. Diurnl hnges in invertse tivity nd solule sugr onentrtio in jsmonte-defiient nd plnts Invertses leve surose into gluose nd frutose following diurnl pttern (Sturm & Tng, 999; N gele et l., ). Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust Higher invertse tivity might therefore led to higher gluose nd frutose pools. To investigte whether the higher gluose nd frutose onentrtio oserved in jsmonte iosynthesis-defiient plnts n e ttriuted to higher invertse tivity, we mesured the tivity of solule nd iolule invertses nd orrelted the rtio of surose (preursor) to gluose nd frutose (produts) with the mesured enzyme tivities. Invertse tivities nd sugr onentrtio were mesured from lef extrts of rosette stge nd plnts t five times of the dy: 7:, :, 3:, 7: nd : h. Five independent replites (plnts) of eh genotype were hrvested per time point. Enzyme tivities (Ferrieri et l., 3) nd sugr onentrtio were mesured s desried y Mhdo et l. (3). Effet of solule sugrs on terpillr growth Low seondry metolite levels re generlly ssumed to e respoile for the inresed lrvl growth of herivores on jsmonte signling-impired plnts (Hlitshke & Bldwin, 3; Rypurm & Bldwin, 6; Pshold et l., 7). To determine whether the higher solule sugr onentrtio in jsmonte-defiient plnts ontriute to the inresed M. sext lrvl growth, we mnipulted sugr onentrtio in plnt nd in vitro nd mesured terpillr growth in five different experiments s follows. Cterpillr growth on sugr-restored, jsmonte iosynthesisdefiient plnts To derese solule sugrs in plnts, we produed hemizygous 9 irrca line y rossing n ira- OC line with n irrca line. Silening RCA slightly impirs photosyntheti tivity in N. ttenut (Mitr & Bldwin, 8), nd we therefore hypothesized tht redution in the photosyntheti pity should redue sugr onentrtio nd, oequently, the hemizygous jsmonte-defiient plnts should hve restored wild-type (WT) sugr onentrtio. To test the vlidity of this ssumption, sugr onentrtio were mesured in the leves of, irrca, nd 9 irrca plnts t 5: h (end of the drk period) nd 3: h (middle of the light period). As M. sext herivory hs een shown to redue sugr New Phytologist (5) 7: 9 5

4 94 Reserh New Phytologist onentrtio in N. ttenut (Mhdo et l., 3), we lso mesured sugr onentrtio fter simulted (wounding nd M. sext orl seretion tretments, W + OS) nd tul (three neontes per plnt for 6 d) M. sext herivory. Intt plnts served s ontrols (n = 5). Mndu sext herivory (W + OS) ws simulted y rolling fri pttern wheel three times on eh side of the midvein of fully developed rosette leves. The wounds were immeditely treted with ll of : 5 (v/v) milliq wter-diluted M. sext orl seretion solution. The tretments were repeted three times every other dy. Following the vlidtion of this in vivo pproh, we determined terpillr growth on the different genotypes. Two M. sext neontes were pled on rosette stge plnts nd llowed to feed freely (n = 5). Seven nd 9 d lter, their mss ws determined using mirolne (Srtorius TE4S; Dt Weighing Systems In., Elk Grove, IL, USA). Mndu sext eggs were derived from n in-house olony nd rered s desried y Grosse-Wilde et l. (). Cterpillr growth on plnts with redued photosynthetilly tive rdition (PAR) As n lterntive me of reduing solule sugrs in plnts, we redued the mount of PAR y overing rosette leves with green filter (Rosolux #443; Roso Lortories In., Stmford, CT, USA). We hypothesized tht reduing PAR supply should redue sugr onentrtio in the leves. Plnts overed with ler filter (Rosolux #; Roso Lortories In.) were used s ontrols. Sugr onentrtio were quntified 3 d fter the strt of the PAR redution tretment s desried y Mhdo et l. (3). Plnts for the sugr mesurements were hrvested t 9: h. Hed spe temperture nd humidity, red to fr-red rtios, strh (Mhdo et l., 3), solule protei (Brdford, 976), verge internode length nd numer of flowers were lso quntified to ssess whether the filters eliited shde voidne respoes nd other seondry effets. To evlute terpillr growth, two M. sext neontes were pled on rosette stge plnts (n = 3) nd llowed to feed freely. Seven nd 9 d lter, lrvl mss ws determined s desried erlier. Cterpillr growth on semi-rtifiil diets Mndu sextindued jsmonte signling depletes solule sugrs nd indues seondry defeive metolites in the leves of N. ttenut plnts; in ontrst with plnts, sugrs re not depleted nd seondry defeive metolites re not indued in respoe to M. sext simulted herivory in jsmonte iosynthesis-defiient plnts (Mhdo et l., 3). To understnd whether the etter M. sext growth on jsmonte-defiient plnts is result of their inresed sugr onentrtion nd/or their deresed levels of seondry metolites, we performed n experiment with semi-rtifiil diets in whih sugrs were omplemented to mth those of WT nd ontrol plnts. The diets were prepred s desried lter, ut the whet germ ws repled with 5 g of dried N. ttenut leves. To generte the neessry plnt mteril, we treted nd plnts with M. sext orl seretio (W + OS indution) s desried y Mhdo et l. (3). These tretments indue plnt defees nd deplete sugrs in the leves of N. ttenut in JA-dependent mnner New Phytologist (5) 7: 9 5 (Mhdo et l., 3). After the tretments, we olleted nd dried the leves (4 h t 5 C). Plnts were hrvested t 3: h. Diets prepred with untreted plnt mteril served s ontrols. Sugr onentrtio in the semi-rtifiil diets were determined, nd susets of diet ues were omplemented with pure sugrs to mth WT nd ontrol levels. Mndu sext growth on the different diets ws then mesured over d. Forty-four neontes per diet type (four lrve per plte; pltes per diet type) were fed d liitum nd the diet ues were repled every other dy. In ddition, terpillr survivorship ws reorded. As sugr omplementtion of plnts my indue seondry respoes (Rollnd et l., 6), the ove pproh llowed us to test the diret ontriution of solule sugrs to herivore growth in plnt mtrix. Cterpillr growth on rtifiil diets enrihed in gluose nd frutose To evlute the individul effet of gluose nd frutose on M. sext growth, we prepred rtifiil diets with different onentrtio of gluose nd/or frutose (see lter, Fig. 7, for tretment omintio) nd mesured M. sext growth. The diets were prepred essentilly s desried y Pohlon & Bldwin () without surose, plnt mteril nd ntiiotis. Briefly, 7 g of gr were dissolved in 5 ml of wter t 5 C nd mixed with 55 g whet germ, g yest extrt, 9 g Wesson slt mixture, 3.5 g sori id,.5 g holesterol,.5 g sori id, 5 ml rw lieed oil,.5 ml formlin nd 9 ml vitmin mixture ( mg niotini id, 5 mg rioflvin, 33.5 mg thimine, 33.5 mg pyridoxine, 33.5 mg foli id nd mg l iotin in wter). The produed food ws liquoted into smll plsti oxes nd kept t 8 C until use. Gluose nd frutose were dissolved in wter nd dded to the diet ues. Diets were freshly prepred nd repled every other dy. Forty terpillrs (four lrve per plte; pltes per diet type) were fed d liitum in limte hmer (45 55% reltive humidity, 4 6 C during dys nd 3 5 C during nights under 6 h of light). Lrvl mss ws determined s desried erlier, 7 nd 9 d fter the eginning of the experiment (n = 4). Intertion etween protein nd solule sugrs The performne of iet herivores depends, mong other ftors, on protein : rohydrte rtios (Ruenheimer et l., 5; Simpson & Ruenheimer, 9; Roeder & Behmer, 4). To investigte whether the oserved negtive effet of inresed dietry solule sugrs on M. sext growth hnges with the mount of ville protein, we prepred rtifiil diets with vritio in sugr nd protein onentrtion nd mesured M. sext lrvl mss nd the mount of ingested diet, nd lulted the effiieny of onversion of ingested food (Wlduer, 968). Diets were prepred ording to Pohlon & Bldwin (). Surose ws repled y inresing onentrtio of gluose nd frutose (, 6 nd mg g of diet) to mimi the tul differenes in solule sugr profiles etween jsmonte iosynthesis-defiient nd WT plnts. To inrese the protein onentrtion of the diets, sein ws dded t onentrtio of 5 or 5 mg g FW. Solule protein onentrtio in plnts estimted y the Brdford method n reh 4.47 mg g in the leves of some plnt speies (Ruiz & Romero, 999). Thirty neontes (three Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust

5 New Phytologist Reserh 95 lrve per plte; pltes per diet type) were fed d liitum nd the ove prmeters were determined 9 d fter the eginning of the experiment (n = 3). Sttistis Unless otherwise stted, sttistil tests were rried out with Sigm Plot. (Systt Softwre In., Sn Jose, CA, USA) using nlysis of vrine. Levene s nd Shpiro Wilk tests were pplied to determine error vrine nd normlity. Holm Sidk nd Dunn s post ho tests were used for pirwise or multiple ompriso. Dtsets from experiments tht did not fulfill the ssumptio for ANOVA were nturl log-, root squre- or rnk-trformed efore nlysis. The effet of semi-rtifiil diets on terpillr survivorship ws nlyzed in R (R Development Core Tem, ) using generlized liner models (GLMs), under qusiinomil distriution with F-test. Residul nlysis ws rried out to verify the suitility of error distriution nd model fitting. Detils on speifi tests rried out in eh experiment re provided in Supporting Informtion Notes S. Results Jsmonte signling negtively ffets lef gluose nd frutose onentrtio Lef solule protein levels of jsmonte pereption-impired ir- COI nd jsmonte iosynthesis-defiient plnts were similr to those oserved in jsmonte-ompetent plnts ross different developmentl stges (Fig. d). Strh did not differ etween genotypes t erly rosette (Fig. e), rosette (Fig. f) nd elongted (Fig. g) stges. Erly flowering ircoi, ut not, plnts ontined higher lef strh onentrtio thn plnts (Fig. h). Gluose nd frutose onentrtio were higher in jsmonte signling-impired plnts ompred with ontrols t erly rosette (Fig. i), elongted (Fig. k) nd erly flowering (Fig. l) stges. At the rosette stge, only frutose onentrtio in plnts were elevted ompred with ontrols (Fig. j). Surose onentrtio did not differ etween genotypes t ny of the evluted developmentl stges (Fig. i l). JA onentrtio re negtively orrelted with lef solule sugrs ross different jsmonte-defiient genotypes Aross eight jsmonte-defiient trgeni lines (Fig. ), we found signifint vritio in solule sugr, JA nd JA-Ile onentrtio (Fig.,). A signifint negtive orreltion etween solule sugr nd otitutive JA onentrtio (P <.) ws oserved (Fig. ). By ontrst, no signifint orreltion etween solule sugr nd JA-Ile onentrtio (P =.3) ws found (Fig. ). Together, the ove experiments demotrte tht jsmontes negtively ffet solule sugr onentrtio in N. ttenut leves. Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust Jsmonte-dependent sugr suppression is orrelted with deresed invertse tivity Over the ourse of the dy, we found tht leves of jsmonte iosynthesis-defiient plnts umulted less surose from 3: to 7: h, ut more gluose nd frutose from : to : h, ompred with ontrols (Fig. 3,). Moreover, solule invertse tivity ws inresed in jsmonte iosynthesis-defiient plnts ompred with plnts from 3: to : h (Fig. 3), n effet whih orrelted negtively with the rtios of surose to frutose nd gluose ross the different smples nd time points (Fig. 3d). By ontrst, iolule invertse tivity ws not ltered y jsmontes nd not orrelted with solule sugr rtios (Fig. 3e,f). Jsmonte-dependent sugr suppression improves herivore weight gin To understnd whether higher lef sugr onentrtio in jsmonte iosynthesis-defiient plnts improve M. sext growth, we redued sugr onentrtio in plnts y silening RCA tivity. A redution in RCA tivity did not ffet solule sugrs in plnts, ut deresed gluose nd frutose onentrtio in plnts (Fig. 4, S). The prtil restortion of WT sugr onentrtio in the 9 irrca rosses ws even more pronouned in herivory-indued plnts (Fig. 4). Surose nd solule protein onentrtio remined lrgely unhnged (Figs 4, S), prt from slight inrese in otitutive surose onentrtio in 9 irrca plnts. From these results, we dedued tht silening RCA prtilly restored WT sugr onentrtio in jsmonte iosynthesis-defiient plnts, nd these lines ould e used to test the influene of solule sugrs on M. sext growth in plnt. Our initil expettion ws tht inresed gluose nd frutose onentrtio would inrese M. sext growth. In ontrst with this hypothesis, we found tht the redued sugr onentrtio in 9 irrca plnts inresed M. sext growth even eyond the highly inresed mss gin in the plnts (Fig. 4j). This result suggests tht jsmonte-dependent sugr depletion redues rther thn enhnes plnt resistne. Mndu sext gi more weight on PAR-limited, sugrdeprived plnts As seond pproh to mnipulte plnt sugr onentrtio, we redued PAR supply y 43% (Fig. 5). As expeted, reduing PAR signifintly redued sugr onentrtio in oth nd plnts (Fig. 5). Although the green filters slightly hnged the red : fr-red rtios (Fig. S3), we found little phenotypi evidene for the tivtion of shde voidne respoes in PAR-redued plnts (Fig. S4,). We lso found no hnges in hed spe temperture nd humidity (Fig. S3,). Strh nd solule protein onentrtio lso remined unltered (Fig. S3,d). Overll, M. sext lrve gined more weight on ira- OC plnts thn on plnts (Fig. 5). PAR redution signifintly inresed terpillr weight gin independent of the New Phytologist (5) 7: 9 5

6 96 Reserh New Phytologist Erly rosette Solule protein Genotype: P =.938 Stge: P =.78 G S: P =.557 Strh Genotype: P =.86 Stge: P <. G S: P =.4 () (e) 7 6 (i) 8 Solule sugrs Genotype: P <. Stge: P <. G S: P <. Rosette Elongted Erly flowering mg g FW () 7 (f) 6 (j) () 7 (g) 6 (k) (d) 7 (h) 6 (l) ircoi ircoi ircoi Jsmonte iosynthesis-defiient Jsmonte pereption-impired Jsmonte-ompetent Surose Frutose Gluose Fig. Jsmonte signling-impired Niotin ttenut plnts umulte higher sugr onentrtio thn jsmonteompetent empty vetor () plnts ross different developmentl stges. Averge ( SE) ( d) solule protein, (e h) strh nd (i l) solule sugr onentrtio of rosette leves t (, e, i) erly rosette, (, f, j) rosette, (, g, k) elongted nd (d, h, l) erly flowering stges. Different letters indite signifint differenes (P <.5, Holm Sidk post ho tests) in totl sugr (gluose, frutose nd surose) onentrtion mong genotypes within developmentl stges. Asterisks indite signifint differenes of eh individul metolite (gluose, frutose or surose) onentrtion mong genotypes ( or ircoi) ompred with within developmentl stges (Holm Sidk post ho tests:, P <.5;, not signifint) (n = 5). plnt s pity to produe jsmontes (Fig. 5). As plnts ontin higher sugr levels, ut lso lower seondry metolites, ompred with plnts, oth ftors potentilly ontriute to the oserved terpillr growth rtes. Mndu sext grows etter on low seondry metolite nd low sugr-ontining semi-rtifiil diets In third pproh, we omplemented semi-rtifiil diets with solule sugrs. This resulted in seven different diets with different sugr onentrtio nd dded plnt mterils (Fig. 6). Sugr omplementtion effetively inresed ontrol sugr New Phytologist (5) 7: 9 5 onentrtio to mth those of ontrol plnts, nd herivory-suppressed sugr onentrtio to mth those of ontrol nd herivory-indued onentrtio (Fig. 6). Mndu sext weight gin did not differ etween lrve tht fed on unindued nd diets (Fig. 6, rs A, C). When sugr onentrtio in unindued diets were omplemented to unindued onentrtio, M. sext growth ws redued (Fig. 6, r B). When feeding on W + OS-indued plnt mteril, M. sext growth ws lower on thn on diet (Fig. 6, rs D, G). When sugr onentrtio of W + OSindued plnt mteril were omplemented to unindued ontrols or to unindued onentrtio, M. sext growth Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust

7 New Phytologist Reserh 97 Fig. Cotitutive jsmoni id nd solule sugr onentrtio re negtively orrelted. () Shemti representtion of the jsmonte signling sde. (, ) Correltion etween verge ( SE) solule sugrs (gluose, frutose nd surose; Glu + Fru + Su) nd () verge ( SE) otitutive jsmoni id levels (JA) or () otitutive jsmonoyl-l-isoleuine (JA-Ile) levels. GLA, glyerolipse A; SIPK, sliyli id-indued protein kie; FACs, ftty id mino id onjugtes; WIPK, woundindued protein kie; AOS, llene oxide synthse; AOC, llene oxide ylse; OPR3, -oxophytodienoi id (OPDA) redutse; OPC-8:, 3-oxo--( -pentenyl)- ylopentne--otnoi id; JAR 4/6, JAmino id synthetse; COI, orontine ieitive ; JAZ, Jsmonte ZIM-domin protein; TF, trription ftors. Metolite nlysis ws rried out in five independent replites of eh Niotin ttenut genotype. () Metolites Memrne lipids Free 8:3 3S-OOH-8:3,3-epoxy-8:3 OPDA OPC-8: JA JA-Ile JA-Ile COI JAZ TF Biosyntheti () enzymes Person s orreltion Regultory 3. r =.888 P <. GLA enzymes SIPK.7 iropr3 Eliitors FACs.3 irsipk irwipk AOS.9 iraos irjar4/6 WIPK irgla AOC OPR3 JA (ng g FW) () Person s orreltion 3. r =.495 P <.3 iropr3 JAR4/6.7 irsipk.3 irwipk iraos TF.9 irjar4/6 JA respoive genes.5 irgla JA-Ile (ng g FW) Glu+Fru+Su (mg g FW) Glu+Fru+Su (mg g FW) ws further redued (Fig. 6, rs E, F). A similr pttern ws oserved for terpillr survivl (Fig. 6). Tken together, these results show tht the W + OS-triggered indution of defee redues M. sext growth nd survivl in jsmonte-dependent mnner, ut tht the jsmonte-dependent W + OS-indued sugr depletion inreses M. sext growth nd therey ompromises indued resistne. Mndu sext grows etter on low sugr-ontining rtifiil diets To disentngle the individul ontriution of gluose nd frutose to M. sext growth suppression, we omplemented rtifiil diets with different omintio of the two sugrs t physiologilly relevnt onentrtio. Mndu sext growth strongly deresed with inresing mounts of gluose nd frutose in dose-dependent mnner, independent of the omintion of sugrs (Fig. 7). On n individul sis, inresed frutose deresed M. sext growth to greter extent thn did inresed gluose (Fig. 7). Exess protein reverses the negtive effet of solule sugrs on M. sext weight gin At protein supply of 5 mg g FW, M. sext grew less well on higher solule sugr diets in dose-dependent mnner (Fig. 8). However, when exess protein t onentrtion of 5 mg g FW ws offered, the opposite effet ws oserved: M. sext now gined more weight on sugr-rih diets (Fig. 8). We lso found tht the mount of ingested diet deresed with inresing sugr onentrtio in protein-independent mnner (Fig. 8). The Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust effiieny of onversion of ingested food did not hnge under norml protein supply, ut tended to inrese with sugr onentrtio under exess protein (Fig. 8). These results demotrte tht the negtive effet of solule sugrs on M. sext growth depends on the protein ontent of the food soure. Under nturl onditio, the mount of ville protein in the leves would result in negtive effet of sugrs on M. sext growth, s protein onentrtio in plnt leves re elow 5 mg g FW. Disussion Our experiments demotrte tht jsmontes inhiit solule invertses nd redue gluose nd frutose onentrtio in N. ttenut leves, nd tht this redution diretly ompromises plnt resistne y inresing M. sext growth nd survivl. Aross different developmentl stges, times of dy, nd trgeni events, jsmonte signling negtively influened the onentrtio of gluose nd frutose in N. ttenut leves, wheres only minor hnges in strh nd surose were found. This suggests tht jsmontes speifilly influene solule monoshride onentrtio. The differenes etween WT nd jsmonte signling-impired plnts were weker t the lte rosette stge thn in younger nd older plnts. It remi to e determined to wht extent this vrition is result of vrition in jsmonte iosynthesis or dowtrem signling. Cotitutive nd indued jsmonte levels re redued in flowering N. ttenut plnts, n effet tht is orrelted with the lower expression of jsmontedependent defees (vn Dm et l., ; Kur et l., ; Diezel et l., ; Onkokesung et l., ). The ft tht jsmonte-dependent sugr regultion did not follow this pttern New Phytologist (5) 7: 9 5

8 98 Reserh New Phytologist mg g FW () : h : h Surose Two-wy ANOVA Genotype: P =.87 Time: P <. G T: P =.68 3: h Time of dy Solule sugrs () 7: h : h mg g FW.5.5 7: h Gluose + Frutose : h 3: h 7: h Time of dy Two-wy ANOVA Genotype: P <. Time: P <. G T:P =.6 : h Invertse tivity µmol Glu g FW min Invertse tivity µmol Glu g FW min () (e) Two-wy ANOVA Genotype: P <. Time: P <. G T: P =. 7: h : h 3: h Time of dy Two-wy ANOVA Genotype: P =.56 Time: P =. G T:.886 7: h : h 3: h 7: h 7: h Time of dy Solule invertses : h (d).5 Su : (Glu + Fru).5 Iolule invertses : h (f).5 Su : (Glu + Fru) Person s orreltion r =.477 P < Invertse tivity (µmol Glu g FW min ) Person s orreltion r =.39 P =.99 Invertse tivity.4 (µmol Glu g FW min ) Fig. 3 Diurnl jsmonte-dependent suppression of solule sugrs is ssoited with deresed invertse tivity. Averge ( SE) () surose nd () gluose nd frutose onentrtio. Averge ( SE) () solule nd (e) iolule invertse tivity. (d, f) Correltion etween the rtio of surose (Su) to gluose (Glu) nd frutose (Fru) nd (d) solule invertse tivity or (f) iolule invertse tivity. Asterisks indite signifint differenes within eh time point (Holm Sidk post ho tests:, P <.5;, P <.;, P <.;, not signifint). Metolite nd enzyme tivity mesurements were rried out in five independent replites (individul Niotin ttenut plnts) for eh genotype nd time point., empty vetor;, llene oxide ylse-silened plnts. suggests role of dowtrem signling, rther thn diret effet of jsmonte iosynthesis, on the oserved developmentl ptter. We found tht the totl solule protein remined otnt in the leves of N. ttenut plnts ross different developmentl stges, times of dy, trgeni events nd plnt tretments. It is importnt to note tht we mesured protein onentrtion y the Brdford method. As this method requires idi onditio, nd s the soluility of RuBisCO, one of the most undnt protei, is deresed under low ph, we my hve underestimted the totl protein onentrtio. However, using 5 N leling nd LC- MS E, it ws demotrted tht investment into RuBisCO iosynthesis is not hnged in jsmonte-defiient inverted repet New Phytologist (5) 7: 9 5 lipoxygee (irlox3) N. ttenut plnts (Ullmnn-Zeunert et l., 3). However, herivore ttk deresed RuBisCO levels in WT nd, leit to lesser extent, irlox3 plnts (Ullmnn- Zeunert et l., 3). A detiled nlysis of the most undnt solule protei in jsmonte signling-impired plnts using similr pprohes might help us to understnd whether nd how jsmonte signling regultes solule protein levels in plnts in more detil. Over the ourse of the dy, we oserved tht defiienies in jsmonte signling inresed gluose nd frutose onentrtio, s well s invertse tivity, in the leves of rosette stge N. ttenut plnts during the light phse. We lso noted slight suppression in surose onentrtio round middy. Bsed on Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust

9 New Phytologist Reserh 99 Solule sugrs (mg g FW) Control W + OS M. sext () () () Gluose (d) (e) (f) Frutose (g) Surose (j) (h) irrca irrca Tretment (i) Lrvl mss (g) irrca irrca A Cterpillr growth Two-wy ANOVA Genotype: P <. Age:P <. G A: P <. B C irrca irrca D 9 d 7 d Gluose Two-wy ANOVA Genotype: P <. Tretment: P <. G T: P =.33 Frutose Two-wy ANOVA Genotype: P <. Tretment: P =. G T: P =.9 Surose Two-wy ANOVA Genotype: P <. Tretment: P =.9 G T: P =.4 () () µmol m s 3 mg g FW 8 4 Two-wy ANOVA Genotype: P <.9 PAR-red.: P <. G P: P =.97 Experimentl set up PAR Norml PAR 43% Norml PAR PAR-redued Solule sugrs PAR-redued Norml PAR PAR-redued Surose Frutose Gluose ().5 Cterpillr growth D Fig. 4 Reduing solule sugr onentrtio through riulose-,5- isphosphte roxylse/oxygee tivse (RCA) inhiition inreses Mndu sext growth. Averge ( SE) (,, ) gluose, (d, e, f) frutose nd (g, h, i) surose onentrtio of (, d, g) ontrol, (, e, h) wounding nd M. sext orl seretion (W + OS)-treted nd (, f, i) M. sextttked plnts hrvested t 3: h. (j) Averge ( SE) mss of M. sext lrve rered on different Niotin ttenut genotypes (n = 35 5). irrca, riulose-,5-isphosphte roxylse/oxygee tivsesilened plnts;, llene oxide ylse-silened plnts; irrca 9 irrca, hemizygous rosses etween AOC- nd RCA-silened plnts;, empty vetor-trformed plnts. Different letters indite signifint differenes (Holm Sidk post ho tests: P <.5) mong genotypes within eh tretment nd metolite for ( i) nd within time point for (j). Asterisks indite signifint differenes mong eh tretment nd ontrol plnts within eh genotype nd metolite (Holm Sidk post ho tests:, P <.). Dshed lines indite the levels of eh metolite for intt plnts. Metolite mesurements were rried out in five independent replites of eh genotype nd tretment. (j) The lower portion represents the weight tht terpillrs rehed fter 7 d, nd the totl height of the rs represents the weight tht terpillrs rehed fter 9 d. Therefore, the upper portion represents only the weight tht terpillrs gined etween dy 7 nd 9. the positive orreltion etween invertse tivities nd preursor to produt rtios of the different sugrs, we propose tht jsmontes might regulte sugr onentrtio through the suppression of invertse tivity. Invertses re well known to ontrol the Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust Lrvl mss (g) A Norml PAR B PAR-redued C Norml PAR PAR-redued 9 d 7 d Two-wy ANOVA 7d Genotype: P <. PAR-red: P =.46 G P: P =.674 9d Genotype: P <. PAR-red: P <. G P: P =.336 Fig. 5 Redution in photosynthetilly tive rdition (PAR) in Niotin ttenut plnts dereses solule sugrs nd inreses Mndu sext growth in jsmonte-independent mnner. () PAR under PAR-redued nd norml glsshouse PAR onditio. () Averge ( SE) sugr onentrtio of N. ttenut plnts under PAR-redued nd norml onditio. () Averge ( SE) mss of 7- nd 9-d-old M. sext lrve feeding on plnts grown under PAR-redued nd norml onditio (n = 36 48). Asterisks in () indite signifint effet of filter tretments on PAR (Holm Sidk post ho tests:, P <.). Asterisks in () indite signifint differenes in sugr onentrtion etween plnts under PAR-redued nd norml PAR onditio within eh genotype (Holm Sidk post ho tests:, P <.5). Different letters indite signifint differenes (Holm Sidk post ho tests: P <.5) mong tretments within terpillr ge. Metolite mesurements were rried out in five independent replites of eh genotype nd tretment., empty vetor;, llene oxide ylse-silened plnts. New Phytologist (5) 7: 9 5

10 Reserh New Phytologist mg g of diet Lrvl mss (g) Cterpillr survivorship (%) () () () A B C D E F G P <. A B C D E F G A B C D E F G P <. Solule sugrs in diet + sugrs Control rtio of gluose nd frutose to surose (Zrenner et l., 996; Ohym & Hiri, 999; Tng et l., 999; Jin et l., 9; Bhskr et l., ). Silening of vuolr invertse gene in potto, Solnum tuerosum, ws found to derese vuolr invertse tivity, inrese surose nd redue gluose nd frutose (Bhskr et l., ). Similrly, Zrenner et l. (996) found positive Cterpillr growth + sugrs + sugrs P <. Cterpillr survivorship OS-treted Surose Frutose Gluose Fig. 6 Solule sugr omplementtion of semi-rtifiil diet redues Mndu sext growth nd survivl. () Averge ( SE) solule sugrs in diets. () Averge ( SE) mss of M. sext lrve rered on semi-rtifiil diets tht differ in their primry nd seondry hemil profiles (n = 38). () Averge ( SE) proportion of living terpillrs t the end vs the eginning of the experiment. Different letters in () nd () indite signifint differenes in totl sugr nd terpillr growth (Dunn s post ho tests: P <.5). Different letters in () indite signifint differenes in terpillr survivl (F-test: P <.5). Upperse letters (A G) serve s guide for the ompriso., empty vetor;, llene oxide ylsesilened plnts. Lrvl mss (g).5. A Cterpillr growth.5 B B B D..5 Gluose (mg g diet) Frutose (mg g diet) orreltion etween the rtio of hexoses to surose nd id-solule invertse tivity in different potto ultivrs. Silening of solule id invertse resulted in lower rtios of hexoses to surose. It is noteworthy to mention tht other studies in poplr nd thle ress hve doumented tht exogenous jsmonte pplition inreses invertse tivity (Arnold & Shultz, ; Bogtek et l., ; Arnold et l., 4; Ferrieri et l., 3; Horie et l., 3). The ft tht otitutive jsmonte defiieny led to higher invertse tivity in N. ttenut leves suggests tht the outome of indued jsmontes on invertse tivity might e determined y their endogenous onentrtio. Although ltertion in invertse tivity n hnge the rtio of gluose nd frutose to surose, the inrese in gluose nd frutose is not lwys proportionl to the derese in surose onentrtion (Tng et l., 999; Bhskr et l., ), inditing tht totl sugr pools might lso e regulted y other ftors, inluding, for exmple, hnges in photosyntheti effiieny, ron ssimiltion, glyolyti tivity, surose synthse tivity, surose trporter tivities nd mehnisms of phloem loding/unloding. Indeed, reduing the tivity of RCA, whih modultes the tivity of RuBisCO, the enzyme tht rries out the first mjor step of CO fixtion in plnts (Rines, 3), in jsmonte iosynthesis-defiient plnts redued gluose nd frutose onentrtio y 54% nd 57%, respetively, suggesting tht jsmonte defiieny might ffet sugr umultion vi the regultion of this enzyme. Although we hve no evidene for hnges in glyolyti enzyme tivity, surose synthse tivity, surose trporter tivities nd mehnisms of phloem loding/ unloding, future studies might investigte their ontriution to the higher umultion of gluose nd frutose in jsmonte signling-impired plnts. Given the importne of jsmontes for plnt herivore intertio, we were interested in understnding whether nd how the jsmonte-dependent redution of solule sugrs influenes the resistne of N. ttenut to M. sext terpillrs. Through four orthogonl lines of evidene, we show tht M. sext growth is redued, rther thn enhned, through inresed dietry C Two- wy ANOVA Type of diet: P <. Age: P <. T A: P <. Fig. 7 Mndu sext grow less well on rtifiil diets ontining higher gluose nd frutose onentrtio. Averge ( SE) mss of 7- nd 9-dold M. sext lrve rered on rtifiil diets ontining vrile mounts of gluose nd frutose (n = 3 39). Different letters indite signifint differenes within time point (Holm Sidk post ho tests: P <.5). 9 d 7 d New Phytologist (5) 7: 9 5 Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust

11 New Phytologist Reserh () Lrvl mss (g) () g DW lrve d () g DW lrve g DW diet Cterpillr growth Two-wy ANOVA Protein: P <. Sugrs: P =.799 P S: P <. rohydrtes, n effet tht is ssoited with redution in the mount of ingested food. First, reduing sugr onentrtio through RCA silening in jsmonte-defiient plnts signifintly improved M. sext growth. plnts re lmost ompletely defiient in mny defeive seondry metolites (Mhdo et l., 3; Frgoso et l., 4). Therefore, this result points to seondry metolite-independent growth effet of solule sugrs on M. sext. The etter growth of M. sext on nonhemizygous irrca plnts hs een ttriuted to the redued levels of seondry metolites, n effet driven y the RCA-medited rediretion of the iotive oxylipin JA-Ile to the intive methyl jsmonte (Mitr & Bldwin, 8, 4). However, it is possile tht this effet is lso the result of redued sugr Amount of ingested diet Effiieny of onversion of ingested diet Two-wy ANOVA Protein: P =.59 Sugrs: P =.6 P S: P =. Two-wy ANOVA Protein: P <. Sugrs: P =. P S: P =. mg g 6 mg g mg g mg g 6 mg g mg g Sugr ontent Sugr ontent Protein ontent Protein ontent 5 mg g 5 mg g Fig. 8 Inresing protein supply eyond nturl onentrtio inverts the effet of sugrs on Mndu sext growth. () Averge ( SE) mss of M. sext lrve rered on rtifiil diets ontining different mounts of protein nd solule sugrs (n = 3 3). () Averge ( SE) mount of ingested diets y M. sext lrve. () Averge ( SE) effiieny of onversion of ingested food. Different letters indite signifint differenes (Holm Sidk post ho tests: P <.5) within type of diet. Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust onentrtio in this line t ertin times of the dy (see Supporting Informtion, Fig. S). Seond, reduing sugrs y limiting PAR supply lso improved M. sext growth independent of the plnt s pity to produe jsmontes. Although PAR redution my hve dditionl effets on plnt physiology, we did not find ny evidene for either the shde voidne respoe or hnges in strh nd protein prodution in plnts growing under PAR-redued onditio. Furthermore, the ft tht we oserved similr PAR effets on terpillr growth in nd plnts llows us to exlude seondry effets on plnt seondry metolites s key determinnts of iet growth (Pshold et l., 7). Third, supplementing miniml rtifiil diets ontining dried plnt mteril with sugrs redued M. sext growth nd survivl. Artifiil diets ontining plnt mteril hve een used in previous studies to ssess the ontriution of jsmonte-indued hnges in plnt metolism to M. sext growth (Pohlon & Bldwin, ). Mndu sext grows less well on rtifiil diets ontining jsmonte-treted N. ttenut plnt mteril thn on diets ontining nontreted plnt mteril, n effet tht is positively orrelted with the jsmonte-dependent indution of protese inhiitors (PIs) nd niotine (Pohlon & Bldwin, ). The ft tht terpillrs grew less well on indued thn plnts onfirms tht this ssy n e used to reprodue nturl resistne ptter, whilst llowing for the omplementtion of semi-rtifiil diet with solule sugrs without the onfounding effet of sugr signling on plnt physiology. Fourth, omplementing rtifiil diets with physiologilly relevnt onentrtio of individul solule sugrs onfirmed their negtive, dose-dependent effet on M. sext growth t physiologilly relevnt protein onentrtio. Crohydrte-rih rtifiil diets hve een shown to redue iet performne (Ruenheimer & Simpson, 997; Lee et l., 3; Ruenheimer et l., 5; Bi et l., 8; Merkx-Jques et l., 8) nd survivl (Ruenheimer et l., 5) in erlier studies, effets tht re ssoited with greter propeity to store the exess of ingested rohydrte s ody ft (Chippindle et l., 996; Simpson et l., 4; Wrrik-Smith et l., 6). Although we did not determine lipid onentrtio in the terpillrs, the ft tht the semi-rtifiil diet experiment showed tht lrvl survivl nd weight gin were positively orrelted indites tht the hevier terpillrs re not neessrily ftter, ut lso fitter. Diret mesurements of ody ft would e neessry to onfirm this hypothesis. The post-ingestion mehnisms tht iets use to ope with n exess of dietry rohydrtes inlude the down-regultion of rohydrte-tolizing enzymes (Kotkr et l., 9; Clissold et l., ), the inrese in respirtion rtes (Znotto et l., 997), the up-regultion of gluose-oxidizing enzymes (Merkx-Jques & Bede, 5) nd the inrese in rohydrte egestion (Telng et l., 3; Bi et l., 8). In ddition to the storge of exess dietry rohydrtes s ody ft, the erlier mentioned mehnisms might result in metoli osts for terpillrs tht potentilly redue their optiml growth nd development. Iet guts host n enormous nd phylogenetilly diverse group of miroorgnisms (Engel & Morn, 3). Although their enefiil roles re inresingly eing reognized (Slem New Phytologist (5) 7: 9 5

12 Reserh New Phytologist et l., 3, 4), they re lso potentilly deleterious (Bsset et l., ; Nehme et l., 7; Buhon et l., 3). Altertion in the gut miroil homeostsis is known to influene iet ehvior (Shron et l., ) nd proly lso ffets iet performne. We hypothesize tht inresing dietry rohydrtes ould negtively impt M. sext growth in two wys. The ingestion of high levels of rohydrtes might inrese the size of miroil ommunities to levels tht first outompete M. sext for limiting nutrients, suh s nitrogen, nd, seond, lter the miroil ommunity homeostsis so s to inrese the prevlene of pthogeni miroorgnisms. Coistent with the first hypothesis, we found tht the negtive effets of ingesting n exess of dietry rohydrtes on M. sext growth were reversed y inresing the mount of dietry protein. It remi to e investigted to wht extent this proess might e driven y hnges in the M. sext gut miroil ommunity. We found tht the effiieny of onversion of ingested food tended to inrese with inresing dietry protein onentrtion nd, s oequene, M. sext might hve een le to ope etter with exess dietry rohydrtes. Protein qulity nd quntity re sujet to oiderle vrition (Bloem & Duffey, 99; Felton, 996), nd protei n intert with plnt seondry hemistry to ffet digestiility (Zuker, 983). Trypsin proteie inhiitors (TPIs) ould lso modulte the effet of dietry protein ontent on tissue digestiility. It is worth mentioning tht lousts hve een shown to perform etter on low-nitrogen plnts (Cese et l., ), inditing speies-speifi respoes to this nutritionl prmeter. An experimentl pproh to mnipulte protein levels in plnt is to trget RuBisCO, one of the most undnt lef protei (Felton, 996; Tiz & Zeiger, 998) nd one of the min dietry protei for herivores (Felton, 996, 5). Mitr & Bldwin (8) redued the trript levels of RuBisCO in N. ttenut y n Agroterium-medited trformtion, resulting in derese in this protein of up to.5- fold. Silening the expression of this gene, together with reduing PAR nd RCA trripts, nd/or sugr supplementtion to semi-rtifiil diets, my llow for etter understnding of the ontriution of protein, rohydrtes nd their rtios to iet performne in plnt seondry hemistry ontext. It is importnt to note tht the negtive effet of sugrs on terpillr growth ws only inverted when protein levels were inresed signifintly eyond those typilly found in leves. We therefore expet the mgnitude, ut not the diretion, of the jsmontedependent sugr depletion effet to hnge with more modest hnges in protein levels. The results of our study re of potentil signifine for the evolution of plnt defee syndromes. Nturl N. ttenut popultio exhiit high phenotypi plstiity in herivory-indued jsmonte prodution (Mhdo et l., 3), whih is positively orrelted with seondry metolite iosynthesis (Gquerel et l., 9) nd might therefore e orrelted with herivore resistne (Royo et l., 999; Hlitshke & Bldwin, 3; Li et l., 4; Kllenh et l., ). We found tht jsmonte defiieny leds to higher sugr onentrtio in leves whih my, in turn, redue M. sext performne. Therefore, this jsmonte-dependent sugr depletion might led to New Phytologist (5) 7: 9 5 trde-offs tht ontriute to nturl vrition in jsmonte signling in nture nd my fvor jsmonte-independent resistne mehnisms. Aknowledgements We would like to thnk the Mx Plnk Soiety for finnil support. We would lso like to thnk the root herivore intertio group, the ITB group memers nd the glsshouse tem for their support. Speil thnks go to Mreike Shirmer nd Melnie Armruster for tehnil support. We thnk three nonymous reviewers for their helpful omments on previous version of the mnusript. M.E. ws supported y Mrie Curie Intr Europen Fellowship (grnt no. 737). C.C.M.A. ws supported y the Brzilin Ntionl Counil for Reserh (CNPq; grnt no. 3799/-). A.P.F ws supported y n Alexnder von Humoldt Postdotorl Fellowship. Referenes Adl G, Cstro G, Miersh O, Pere D.. Chnges in jsmonte nd gierellin levels during development of potto plnts (Solnum tuerosum). Plnt Growth Regultion 36: 6. Appel HM, Arnold TM, Shultz JC.. Effets of jsmoni id, rnhing nd girdling on ron nd nitrogen trport in poplr. New Phytologist 95: Arnold T, Appel H, Ptel V, Stoum E, Kvlier A, Shultz J. 4. Crohydrte trlotion determines the phenoli ontent of Populus folige: test of the sink soure model of plnt defee. New Phytologist 64: Arnold TM, Shultz JC.. Indued sink strength s prerequisite for indued tnnin iosynthesis in developing leves of Populus. Oeologi 3: Bi B, Poisson A, Drwish S, Lsse J, Merkx-Jques M, Desplnd E, Bede JC. 8. Influene of dietry nutritionl omposition on terpillr slivry enzyme tivity. Journl of Iet Physiology 54: Bst BA, Ferrieri RA, Gry DW, Lerdu M, Shlyer DJ, Shueller M, Thorpe MR, Ori CM. 5. Jsmoni id indues rpid hnges in ron trport nd prtitioning in Populus. New Phytologist 67: Bsset A, Khush RS, Brun A, Grdn L, Bord F, Hoffmnn JA, Lemitre B.. 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Reveling omplexity nd speifiity in the tivtion of lipse-medited oxylipin iosynthesis: speifi role of the Niotin ttenut GLA lipse in the tivtion of jsmoni id iosynthesis in leves nd roots. Plnt, Cell & Environment 34: Brdford MM A rpid nd seitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein dye inding. Anlytil Biohemistry 7: Ó 5 The Authors New Phytologist Ó 5 New Phytologist Trust