Endogenous hormone concentrations correlate with fructan metabolism throughout the phenological cycle in Chrysolaena obovata

Transcription

1 nnls of otny Pge 1 of 13 doi:1.193/o/mv53, ville online t Eogenous hormone onentrtions orrelte with frutn metolism throughout the phenologil yle in Chrysolen oovt thos Poli Rigui 1,2, Mríli Gspr 1, Vness F. Oliveir 1,3, Edurdo Purgtto 4 Mri ngel Mhdo de Crvlho 1, * 1 Núleo de Pesquis em Fisiologi e ioquími, Instituto de otâni, PO ox 6841, CEP , São Pulo, SP, rzil, 2 Progrm de Pós-Grdução em iodiversidde Vegetl e Meio miente, Instituto de otâni, São Pulo, rzil, 3 Universidde de Mogi ds Cruzes Cmpus Vill Loos, CEP 535-, São Pulo, SP, rzil 4 Deprtmento de limentos e Nutrição Experimentl/NPN, Fuldde de Ciênis Frmêutis, Universidde de São Pulo, CEP São Pulo, SP, rzil * For orrespoene. E-mil mm.rvlho@gmil.om Reeived: 1 Deemer 214 Returned for revision: 16 Ferury 215 epted: 2 Mrh 215 kgrou ims Chrysolen oovt, n steree of the rzilin Cerrdo, presents sesonl growth, mrked y senesene of eril orgns in winter susequent regrowth t the e of this seson. The uergrou reserve orgns, the rhizophores, umulte inulin-type frutns, whih re known to onfer tolerne to drought low temperture. Frutns frutn-metolizing enzymes show hrteristi sptil temporl distriution in the rhizophores during the developmentl yle. Previous studies hve shown orreltions etween sisi id () or iole eti id (I), frutns, dormny tolerne to drought old, ut the signlling mehnism for the eginning of dormny sprouting in this speies is still unknown. Methods dult plnts were smpled from the field ross phenologil phses inluding dormny, sprouting vegettive growth. Eogenous onentrtions of I were determined y GC-MS-SIM (gs hromtogrphy mss spetrometry seleted ion monitoring), mesurements were mde of frutn ontent omposition, enzyme tivities. The reltive expression of orrespoing genes during dormny sprouting were lso determined. Key Results Plnts showed high frutn 1-exohydrolse (EC ) tivity expression during sprouting in proximl segments of the rhizophores, iiting moiliztion of frutn reserves, when onentrtions were reltively low preipittion temperture were t their minimum vlues. Conomitntly, higher I onentrtions were onsistent with the role of this regultor in promoting ell elongtion plnt growth. With high rtes of preipittion high tempertures in summer, the frutn-synthesizing enzyme surose:surose 1- frutosyltrnsferse (EC ) showed higher tivity expression in distl segments of the rhizophores, whih deresed over the ourse of the vegettive stge when onentrtions were higher, possily signlling the entry into dormny. Conlusions The results show tht frutn metolism orreltes well with eogenous hormone onentrtions environmentl hnges, suggesting tht the o-ordinted tion of rohydrte metolism hormone synthesis enles C. oovt to survive unfvourle field oitions. Eogenous hormone onentrtions seem to e relted to regultion of frutn metolism to the trnsition etween phenophses, signlling for energy storge, reserve moiliztion umultion of oligoshrides s osmolytes. Key words:, I, frutn metolism, Chrysolen oovt, Vernoni here, steree, inulin, hormone signlling, rohydrte reserves, rhizophore, Cerrdo, sesonl dormny. INTRODUCTION The Cerrdo is tropil svnn tht overs out 23 % of the entrl region of rzil, eing surpssed in re only y the mzon Rin Forest (Rtter et l., 1997). This iome hosts high iodiversity is onsidered one of the world hotspots (Simon et l., 29). The Cerrdo soil is deep, porous, permele, ontins mixture of s ly with overll low wter retention, poor in nutrients id, due prtly to high levels of l 3þ (Eiten, 1972). The limte is hrterized y mrked dry winter (3 6 months) wet summer, presenting n verge preipittion of 8 2 mm verge nnul tempertures rnging etween C (Dis, 1992). Wter restrition the wide temperture rnge re some of the seletive pressures ting on Cerrdo speies. In this iome, plnts developed dptive strtegies to ope with sesonl wter defiit temperture stress, inluding enlrged uergrou orgns, thik orky rk, hrd slerophyllous leves, lef senesene in the dry seson (Rtter et l., 1997). Sesonl growth reprodutive ptterns re lso typil relted to the limti sesonlity of the Cerrdo, s well s of other tropil svnns (Willims et l., 1997; tlh VC The uthor 215. Pulished y Oxford University Press on ehlf of the nnls of otny Compny. ll rights reserved. For Permissions, plese emil: journls.permissions@oup.om

2 Pge 2 of 13 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Mntovni, 2). Metoli djustments, inluding prodution of omptile solutes tivtion of the ntioxidnt system, omplete the multiple plnt stress response mehnisms in Cerrdo speies. Frutns re solule, osmotilly tive rohydrtes thought to t on memrne stiliztion y iing to holine phosphte groups, inresing tolerne to drought low temperture (Pilon-Smits et l., 1995; Crvlho et l., 27; Hinh et l., 27; Kwkmi et l., 28; Vlluru Vn den Ee, 28; Gri et l., 211). Frutns re widely distriuted in hereous speies of the Cerrdo, showing hnges in ontents omposition ssoited with the phenologil yle, exposure to low temperture, wter vilility other environmentl ftors previling in this iome (Crvlho Dietrih, 1993; Isejim Figueiredo-Rieiro, 1993; Vieir et l., 1995; Portes et l., 28; Gri et l., 211; Oliveir et l.,213). Frutns hve een regrded s seo to strh in importne s storge rohydrtes, eing present in pprox. 15 % of the ontemporry ngiosperms (Hery Wlle, 1993). Speies ontining frutns re distriuted within diverse rnge of fmilies inluding Poee steree. The diversity of distriution the ourrene of frutns mong highly evolved fmilies iite tht genes for frutn metolism in ngiosperms my hve risen in response to one or few seletive pressures in the reltively reent pst (Hery, 1987). Chrysolen oovt (Less.) Demtt., previously nmed Vernoni here (Vell.) Rusy, elongs to the steree fmily, is hereous, perennil is ntive to the Cerrdo. It umultes inulin-type frutns in its uergrou reserve orgns, the rhizophores. Inulin synthesis in C. oovt ours predominntly in younger tissues of rhizophores (distl regions) y the tion of the enzymes surose:surose 1-frutosyltrnsferse (1-SST; EC ), tht tlyses the formtion of the trishride 1-kestose, frutn:frutn 1-frutosyltrnsferse (1-FFT; EC ) tht is involved in oth the inrese derese of frutn hin size. Frutn depolymeriztion ours minly in segments of the rhizophores ner uds tht originte new shoots (proximl regions), y the tion of frutn 1-exohydrolse (1-FEH; EC ) (Portes Crvlho, 26; Portes et l., 28; seg et l., 211). The gene oding for 1-FEH in C. oovt ws isolted its tivity in inulin hydrolysis ws onfirmed y heterologous expression in the Pihi pstoris system (seg et l.,28). Frutn metolism seems to e tightly orrelted with the phenologil yle of C. oovt in ssoition with limti sesonlity. Sprouting of these plnts in spring is followed y vegettive growth flower development. Vegettive growth lsts until erly utumn, when the eril orgns senese go through sission. From this stge on, plnts remin dormnt until the e of winter, when uds sprout to develop new eril orgns. derese in frutn ontents during sprouting iites reserve moiliztion to provide sustrte for growth, followed y n inrese in frutn ontents during the vegettive growth phse (Crvlho Dietrih, 1993). Sugr metolism is uer hormonl ontrol, sisi id () is known to regulte key enzymes trnsript umultion in different rohydrte synthesis pthwys (Trouverie et l., 23; Yng et l., 24). Different phytohormones re known to regulte frutn metolism (Vn den Ee et l., 22). In the promoter region of the gene 1-FEH II isolted from Cihorium intyus, Mihiels et l. (24) identified motifs for response to uxin,, ethylene, gierellins (Gs) sliyli id, reveling the omplexity of the regultion of frutn metolism genes. In llium ep, Chope et l. (26) deteted the onomitnt derese in frutn ontents in uls stored t low temperture, reported the onset of sprouting t ritil minimum onentrtion of eogenous, ssoited with rohydrte moiliztion. In Hordeum vulgre, nother frutn-umulting speies, plnt limtion to old ourred due to onomitnt inrese in solule sugrs onentrtions (rvo et l.,1998). Yng et l. (24) reported tht the onentrtion inresed in whet stems uer drought, orrelted with inreses in surose-phosphte synthse (EC ) 1-FEH, redution in 1-SST. Plnts treted with n inhiitor of synthesis showed redution of surose synthesis in the tivities of 1-FEH id invertse (EC ). n opposite response ws deteted when plnts were treted with exogenous. Further ler evidene of the regultion of frutn metolism y ws presented y Ruusk et l. (28). Using iividul whet ulms fed with, the uthors showed inresed 1-FEH 6-FEH trnsript umultion inhiition of 1-SST, 6-SFT 1-FFT gene expression. Trnsriptome nlysis of ridopsis thlin rie treted with exogenous showed iution of % of the genes, respetively (Seki et l., 22; Rni et l., 23). In ft, Seki et l.(22)reported tht rohydrte metolism is mong the most ffeted y tretment in. thlin. Despite the importne of uxin in the regultion of plnt development, there re only few studies looking t the ssoition of frutn metolism eogenous uxin onentrtions. rreto et l. (21) reported n inrese in frutn onentrtions in plnts of gve tequiln in the presene of 1-nphthleneeti id. However, Trevisn et l. (214) suggested tht uxin ould t s negtive regultor of frutn-synthesizing enzymes in in vitro plnts of C. oovt.more reently, Zhng et l. (214) fou single nuleotide muttion in the promoter region of the whet 1-FEH w3 gene, loted t n uxin response element (RE). The uthors suggest tht this muttion in RE ould ffet 1-FEH gene expression enzyme tivity, ffeting whet stem frutn remoiliztion to grin in different genotypes. Mjor hnges in phytohormone onentrtions our during the phenologil yle of severl speies. Zho et l. (213) pointed out tht environmentl ftors n regulte plnt hormone onentrtions determine the physiologil stte of plnt development. Mdm rodri (214), studying wter use strtegies of five phylogenetilly distnt speies ohiting in the monsoonl tropis of northern ustrli, region hrterized y mrked sesonl vrition in preipittion, fou different ptterns of sensing, synthesis metolism in leves over the ourse of the yer. ording to the uthors, the ontrsting strtegies fou mong the studied speies represent n evolutionry pthwy for dpttion in plnt wter use. lthough orreltion etween phytohormones, frutn metolism tolerne to drought old hs een shown in some studies, the implitions of eogenous hormonl

3 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Pge 3 of 13 hnges in response to wter old stress in nture t different phenologil phses re poorly uerstood. Considering tht C. oovt is n exellent model for studying the regultion of frutn metolism y environmentl ftors, this study imed to evlute eogenous onentrtions of uxin during the trnsition to the dormnt phse (dry seson) to the sprouting phse (wet seson), in order to orrelte hormone onentrtions frutn metolism in the rhizophore, s the sink soure orgn, in plnts growing in the Cerrdo. Plnt mteril MTERILS ND METHODS Four dult plnts of Chrysolen oovt (Less.) Demtt. were olleted in preserved Cerrdo re t the Reserv iológi eestção Experimentl de Mogi Guçu, SP, rzil (22 18 S, W) t eh of the following phenologil phses: one hrvest t erly dormny (ED) (low wter vilility), three hrvests during sprouting, from erly (S1) through two more dvned stges (S2, S3), two hrvests during the vegettive growth phse (V1, V2) (high wter vilility) one hrvest t dormny (D) (low wter vilility), totlling seven smpling dtes (Tle 1). t eh hrvest, smples of the eril orgns (stems leves) t the sme developmentl stge were used for dry mss determintion hormone nlyses. Rhizophores were wshed immeditely fter hrvest smple frgments were ut from the distl proximl regions, s desried (see fig. 1 in Portes Crvlho, 26; Supplementry Dt Fig. S1). Smples from the two regions were weighed frozen in liquid nitrogen for iohemil moleulr nlyses. Environmentl oitions Meteorologil dt were provided y two ompnies loted ner the iologil Reserve where the study ws rried out. Mximum minimum tempertures were otined from Interntionl Pper do rsil LTD. (22 22 S, W) rinfll dt from two wether sttions in the nery re, elonging to ES Tietê S/ (22 22 S, W) (Fig. 1). The highest preipittion period ourred in summer, etween Deemer 212 Jnury 213, the lowest ourred etween the e of utumn winter, from My to Septemer 212 from pril to July 213, when the temperture rnge TLE 1. Hrvesting dtes orrespoing phenologil phses of plnts of Chrysolen oovt t Reserv iológi e Estção Experimentl de Mogi Guçu, SP, rzil Dte Phenologil phse revition 26 June 212 Erly dormny ED 14 ugust 212 Sprouting S1 11 Septemer 212 Sprouting S2 5 Otoer 212 Sprouting S3 16 Jnury 213 Vegettive V1 17 pril 213 Vegettive V2 1 July 213 Dormny D inresed (Fig. 1). The verge temperture the umulted preipittion during the 7 d preeding eh smpling dte were lulted (Fig. 1). Preipittion ws lose to zero etween ugust Otoer (sprouting stge). The highest preipittion vlues were oserved in Jnury (vegettive stge). The highest temperture rnge ws registered in Otoer, the lowest in Jnury. Soil smples were olleted t the hrvest re in triplite t depths of 1, m, for estimtion of soil wter retention urves (Cruz et l., 25) t Instituto gronômio de Cmpins (IC), SP, rzil. For soil wter retention urves, see Supplementry Dt Fig. S2. Wter sttus of soil plnts Soil moisture ws mesured grvimetrilly in soil smples otined from the plnt rhizosphere (lke, 1965). Wter ontent (WC) of eril orgns rhizophore segments were determined in four smples t eh phenologil phse, using the eqution WC ¼ (f. wt d. wt)/f. wt 1, where f. wt is the fresh weight d. wt the dry weight of the smple. The d. wt of eril orgns ws otined fter drying tissues t 6 Cto onstnt weight, the d. wt of rhizophores ws otined fter smple lyophiliztion. The wter potentil of rhizophore segments ws determined in four smples, in squeezed ell sp, using vpour pressure osmometer (Model VPRO 552, Wesor, Logn, UT, US), ording to Oliveir et l. (213). Osmolrity ws mesured in mmol kg 1 trnsformed into MP, using the Vn t Hoff eqution, MP ¼ mmol kg (Snt-Cruz et l., 22). Determintion of eogenous onentrtions of I y gs hromtogrphy mss spetrometry seleted ion monitoring (GC-MS-SIM) The extrtion ws done s desried in Ludwig-Muller et l. (28) using frozen smples of eril orgns rhizophores grou in mortr, in liquid N 2. n isopropnol:eti id (95:5) solution ws dded to the tissue powder (4:1 v/w) together with 5 lg of eh lelled strd ([ 2 H 6 ]) iole eti id (I) strd ([ 13 C 6 ]I). The mixture ws kept uer shking t 4 Cfor2hentrifuged t 13 g. The superntnt ws olleted onentrted to 5 ll uer n N 2 gs flow, followed y the ddition of 2 ml of ultrpure wter. Smples were then frtionted with 5 ml of ethyl ette the orgni phse ws trnsferred to nother tue dried uer N 2 gs flow. The mteril ws methylted with dizomethne, dried uer N 2 gs flow re-suspeed in ethyl ette. The nlysis ws performed on Hewlett-Pkrd (Wilmington, DE, US) gs hromtogrph model 689 oupled to mss spetrometer model 5973 in seletive ion monitoring mode (GC-MS-SIM). The hromtogrph ws equipped with n HP-171 olumn (3 m, ID 25 mm, 5 lm thik internl film) using helium s the rrier gs t flow rte of 4 ml min 1 in the following progrm: 3 min t 15 C, followed rmp y 5 Cmin 1 to 21 C 15 Cmin 1 to 26 C. Ions with mss rtio/ hrge (m/z) of (orrespoing to eogenous

4 Pge 4 of 13 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Preipittion (mm) Preipittion (mm) M J J S O N D J F M M J J Months Preipittion Mx. temperture Men preipittion Totl preipittion Mx. temperture Men temperture Min. temperture Men temperture Min. temperture ED (Jun) S1 (ug) S2 (Sep) S3 (Ot) V1 (Jn) V2 (pr) D (Jul) Phenologil phses Temperture ( C) Temperture ( C) FIG. 1. () Monthly verge preipittion, monthly men, mximum minimum tempertures etween My 212 July 213 in the study re. () verge umulted preipittion, men, mximum minimum tempertures 7 d efore the hrvest dtes. I) (orrespoing to [ 13 C 6 ]I), lso ions with (m/z) 134, (orrespoing to eogenous ) 138, (orrespoing to [ 2 H 6 ]) were monitored. Conentrtions were lulted sed on extrted hromtogrms t m/z for I for. The extrtions were performed in triplite. Enzyme extrtion ssys Frozen rhizophore smples were pulverized in liquid N 2 homogenized in 5 M MIlvine uffer (ph 55); (1:3, w/v) ontining 2 mm EDT, 2 mm -merptoethnol, 5 mm sori id 1 % polyvinylpolypyrrolidone (PVPP; w/w) s in seg Crvlho, (24). Proteins were preipitted with (NH 4 ) 2 SO 4 t 8 % sturtion, re-suspeed in the rtio of pprox. 1 g fresh mss equivlent per ml in extrtion uffer. The finl extrt ws used for enzyme ssys protein determintion (rdford, 1976), using ovine serum lumin s strd. Enzymes were ssyed y inution of the protein extrt with different sustrtes mixed 1:1 (v/v). Sustrtes were prepred in 5 M MIlvine uffer (ph 45 for 1-FEH, ph 55 for 1-SST 1-FFT). The extrts were inuted t 3 C t finl onentrtion of 2 M surose (Sigm- ldrih) for 1-SST, 2 M 1-kestose for 1-FFT 5 % inulin from C. oovt for 1-FEH tivity. Inution time ws 1 h for 1-SST 1-FEH, 2 h for 1-FFT. For determintion of 1-SST, 1-FFT 1-FEH tivities, smples of the retion mixtures were diluted nlysed using high-performne nion exhnge hromtogrphy with pulsed mperometri detetion (HPEC/PD) with 2 25 mm CroP P-1 olumn on Dionex system (model ICS 3, Dionex, Sunnyvle, C, US), s desried in Oliveir et l. (213). The tivities of 1-FEH, 1-SST 1-FFT were lulted y diret mesurement of frutose, 1-kestose nystose, respetively, using the externl strd method. ll proedures were done t 5 C extrtions were performed in triplite. Solule rohydrte nlyses Solule rohydrtes were extrted from freeze-dried rhizophores (Portes Crvlho, 26). The rude extrt ws sumitted to ethnol preipittion the fruto-oligoshride [hexoses, surose frutns with degree of polymeriztion (DP) 3 27] fruto-polyshride (minly frutns with DP 1 6) frtions were seprted y entrifugtion. Free omined frutose ws mesured in rude extrts in the two frtions seprtely, using ketose-speifi modifition of the nthrone retion frutose (Sigm-ldrih) s

5 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Pge 5 of 13 strd (Jermyn, 1956), the fruto-oligo:frutopolyshride rtio ws lulted. Solule rohydrtes in rude extrts were de-ionized through ion exhnge olumns (Crvlho Dietrih, 1993), nlysed using HPEC/ PD with the sme oitions s desried ove for 1-FEH tivity nlysis. Extrtions were performed in qudruplite. Totl RN extrtion gene expression nlysis Totl RN ws isolted from the proximl distl regions of rhizophores from plnts in sprouting (S1 S3), vegettive (V1) dormnt (D) phses, using n RNesy Plnt Mini Kit (Qigen). For DN synthesis, duplites of 1 lg of totl RN from eh smple were treted with DNse I, RNse-free (Thermo Sientifi) then reverse trnsried using rom hexmers Revertid H Minus Str DN Synthesis (Thermo Sientifi). The quntittive rel-time PCRs (qrt-pcrs) were performed in tehnil triplites using the regent EXPRESS SYR GreenER qpcr SuperMix (Invitrogen) in thermoyler Msteryler VR ep relplex 2S (Eppeorf G, Hmurg, Germny) with the following progrm: 2 min t 5 C, 2 min t 95 C followed y 4 yles of 15 s t 94 C, 1min t 6 C. For the melting urve, the temperture ws inresed grdully over 2 min, from 6 to 95 C. The mrn expression vlues were determined ording to Pfffl (21), reltive to the vegettive phse (ontrol). Reltive gene expression nlysis ws performed for 1-FEH 1-SST genes, expression levels were normlized to the referene genes elongtion ftor (EF) 18S rrn (18S), whih were rnked s the most stle referene genes for gene expression nlysis in C. intyus (Mroufi et l., 21). For primer detils, see Supplementry Dt Tle S1. The primer speifiity ws vlidted y melting urves showing mplifition of single produt. PCR effiienies vried from 93 to 99 %. Results were otined from two iologil replites duplite of the reverse trnsription step, s suggested y ustin et l. (29). Sttistil nlyses The orreltion oeffiients were lulted etween edpholimti, iohemil physiologil prmeters temperture, preipittion, soil moisture, wter ontent of plnts, wter potentil of rhizophores, phytohormone solule rohydrte ontents, enzyme tivities using Person s orreltion. Sttistil signifine of results ws tested using iepeent Student s t-tests. One-wy nlyses of vrine (NOVs) were performed using the softwre SISVR 42for omprison of the mens over the smpling period. When signifint differenes were fou, post-ho Tukey s honestly signifintly different (HSD) test ws lso performed t P 5. Dt of distl proximl rhizophore segments were lso ompred t eh smpling dte with one-wy NOV. Prinipl omponent nlysis (PC) ws rried out using the following prmeters: verge preipittion temperture, wter ontent in eril orgns, wter ontent wter potentil of rhizophores, iohemil physiologil prmeters. PC ws performed from the orreltion mtrix with dt trnsformed y logrithm [(log (x þ 1)]. The romiztion test (999 permuttions) ws used to hoose the PC interprettion dimension (P < 5) using the progrm PC-ORD 6.. Wter sttus of soil plnts RESULTS In ordne with the highest umulted preipittion shown in Fig. 1, soil moisture ws highest in the vegettive phse (V1) (Fig. 2). t erly dormny (ED), the highest wter potentil ws oserved ( 52 MP in proximl 6 MP in distl segments), while in sprouting (S1 S3) vegettive (V2) phses, the lowest vlues were deteted (etween MP in oth segments), without mrked differenes etween distl proximl segments (Fig. 2). Wter ontent in the eril orgns remined onstnt during sprouting vegettive phses, differing signifintly only t ED. In S1 dormny (D), the plnts presented no eril orgns (Fig. 2C). In rhizophores, the wter ontent presented smll vritions throughout the nlysed period remined higher in the proximl segment, in omprison with the distl ones, with signifint differenes t ED, S1 D (Fig. 2D). Eogenous onentrtions of I The ontent in the eril orgns teed to inrese, from 58 ng g 1 d. wt in S2 to 3868 ng g 1 d. wt in V2, with the lowest ontent (22 ng g 1 d. wt) t ED (Fig. 3). Stges S1 D were not smpled due to lk of eril orgns. In rhizophores, no vrition in ontents etween proximl distl segments ourred, exept t D, when the distl segment presented vlue signifintly higher thn the proximl one. The highest onentrtions were fou in the proximl segment t V2 (331 ng g 1 d. wt), the lowest, during sprouting (S1 S2), with vlues ner zero (Fig. 3). n opposite profile ws oserved for I ontents in eril orgns; the vlue deteted t V2 (11 ng g 1 d. wt), ws twie s low s in S2 (28 ng g 1 d. wt) (Fig. 3C), lthough no sttistil differenes were deteted. I in rhizophores showed no signifint differenes etween the two segments, the lowest vlue ws deteted in S2 (2 ng g 1 d. wt in the distl segment; Fig. 3D). Solule rohydrtes Fruto-polyshride ontents did not vry signifintly mong the proximl segments during the nlysed period. mong the distl segments, differene ws oserved only etween S3 D. In the ltter, the ontent in the distl segements ws signifintly higher thn in the proximl segment (Fig. 4). Conversely, fruto-oligoshride ontents did not differ etween the rhizophores segments; however, vlues differed mong phenologil phses, with the lowest vlues smpled during sprouting (Fig. 4). The shrp redution of fruto-oligoshrides during sprouting is evidened y the fruto-oligo:fruto-polyshrides

6 Pge 6 of 13 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt y w Rhizophores (MP) Soil moisture (%) Wter ontent of eril orgns (%) Wter ontent of rhizophores (%) C D * Proximl Distl * * Phenologil phses * Proximl Distl ED S1 S2 S3 V1 V2 D FIG. 2. () Soil moisture ontent, () wter potentil (Ww) in proximl distl rhizophore segments (s iited in the key), (C) wter ontent of eril orgns (D) wter ontent of proximl distl rhizophore segments of plnts of Chrysolen oovt t different phenologil phses. Vlues re the mens 6 s.e. (n ¼ 4). Upper se letters iite omprison of proximl segments lower se letters iite omprison of distl segments during the experimentl period (P < 5). n sterisk (*) denotes omprison of the segments (proximl distl) within eh hrvest dte (P < 5)., no dt. * in eril orgns (ng g 1 d. wt) in rhizophores (ng g 1 d. wt) I in eril orgns (ng g 1 d. wt) I in rhizophores (ng g 1 d. wt) C D Phenologil phses rtio, the inrese in this rtio in proximl segments t D ws due to redution of fruto-polyshrides (Fig. 4C). Reduing sugrs showed deresing teeny from ED to S2, followed y n inrese t S3, further derese until D(Fig. 4D). These results were lso oserved in the hromtogrphi profiles, with n inrese of the hexoses gluose frutose during sprouting, espeilly t S3 (Fig. 5). In the ED S1 S2 S3 Proximl Distl Proximl Distl V1 V2 D FIG. 3. () ontent in the eril orgns () in the proximl distl rhizophore segments (s iited in the key), (C) I ontent in the eril orgns (D) in the proximl distl rhizophore segments of plnts of Chrysolen oovt t different phenologil phses. Vlues re the mens 6 s.e. (n ¼ 3). Upper se letters iite omprison of proximl segments lower se letters iite omprison of distl segments during the experimentl period (P < 5). n sterisk (*) denotes omprison of the segments (proximl distl) within eh hrvest dte (P < 5)., no dt.

7 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Pge 7 of 13 Fruto-polyshrides (mg g 1 d. wt) Fruto-oligoshrides (mg g 1 d. wt) Fruto-oligo : frutopolyshride Reduing sugrs (mg g 1 d. wt) C D proximl segment, following V1 until D, grdul inrese in frutns with medium DP (5 15) ws oserved. In the distl segment, n inrese in the 1-kestose pek ws deteted only t V1. Proximl Distl Phenologil phses * ED S1 S2 S3 V1 V2 D FIG. 4. () Fruto-polyshrides, () fruto-oligoshrides, (C) fruto-oligo:fruto-polyshrides rtio reduing sugrs (D) in the proximl distl rhizophore segments (see key) of plnts of Chrysolen oovt t different phenologil phses. Upper se letters iite omprison of proximl segments lower se letters iite omprison of distl segments during the experimentl period (P < 5). n sterisk (*) denotes omprison of the segments (proximl distl) within eh hrvest dte (P < 5). Enzyme tivities gene expression of frutn metolism The tivity of 1-FEH ws highest in the proximl segments of rhizophores throughout the experimentl period, with signifint differenes etween the two segments in S1, V1 V2. Moreover, the overll tivities were higher during sprouting (Fig. 6). The 1-FEH gene showed higher expression during sprouting, eing five times more gretly expressed in the proximl segment t S3 (Fig. 7). The 1-FEH trnsript une showed moderte inrese in distl segments, twie s high s t the vegettive stge (V1), used s the referene (Fig. 7). In ontrst, 1-SST showed the lowest tivities during sprouting ws most tive in the distl segments t ED V1 (Fig. 6). The 1-SST gene showed the highest expression in V1, with mrked inhiition t S1, S3 D (Fig. 7). The enzyme 1-FFT remined tive through ll the phenologil phses nlysed, with mrked derese t S3 higher tivity t ED, S1 S2 (Fig. 6C). The tivity ws signifintly higher in the distl segment only t S1. Prinipl omponent nlysis Prinipl omponent nlysis summrized 552%of the totl dt vriility on the two first xes (Fig. 8), whih were onsidered signifint t the romiztion test (P < 5). ll the smplings t sprouting, exept for the distl segments t S2, t D were positioned on the positive side of xis 1, whih iited orreltion with rhizophore wter ontent (r > 5) 1-FEH tivity (1-FEH) (r > 7). Conversely, on the negtive side of xis 1, distl segments t S2, distl segments t ED ll the smplings of the vegettive phse were orrelted with verge temperture (T) (r > 6), verge preipittion (P) (r > 58), wter ontent (H 2 O O), ([] O) I ([I] O) onentrtions in eril orgns 1-SST tivity (1-SST) (r > 7). Conerning xis 2, the proximl segments t the vegettive phses, ll smplings of S2 S3 were orrelted with I onentrtions in eril orgns (r > 58) verge temperture (r > 6), lso wter ontent in eril orgns (r > 5), while ED D were orrelted with fruto-oligoshride ontents (r > 6), verge preipittion (r > 6). Consequently, the PC xes showed the reltionship of environmentl eogenous vritions the phenologil phses of C. oovt. DISCUSSION This work desries for the first time hnges in frutn metolism in eogenous onentrtions of I ssoited with limti oitions wter sttus during the developmentl yle of Cerrdo speies growing nturlly in the field. We hypothesized tht eogenous onentrtions of hormones hve regultory role in determining the trnsition etween different phenologil phses in the iution of hnges in frutn metolism uer field oitions. Chnges oserved t ED suggest n inrese in sink strength of the rhizophore t this stge, evidened y the inrese in 1-SST 1-FFT tivities in fruto-oligoshrides, minly in distl segments. Sine the reserve tissues umulte high frutn ontents, the inrese in I in these tissues might e n importnt ftor for ell expnsion, neessry for the

8 Pge 8 of 13 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Detetor response (nc) Proximl Distl 34 Erly dormny 27 G 2 13 F S >4 N S >4 6 1-K GF 1-K N Sprouting Sprouting Vegettive Vegettive Dormny Retention time (min) Retention time (min) FIG. 5. HPEC/PD profiles of totl frutns of proximl (left) distl (right) rhizophore segments of plnts of Chrysolen oovt t different phenologil phses. G, gluose; F, frutose; S, surose; 1-K, 1-kestose; N, nystose; >4, frutns with DP higher thn 4. umultion of reserve ompous. t this stge, photossimiltes from the senesing eril orgns re trnsloted to the uergrou reserve orgn used s sustrte for frutn synthesis in similr wy to tht reported for plnts of the sme speies with fully estlished eril orgns (Portes Crvlho, 26) or uer high tmospheri CO 2 onentrtion (Oliveir et l., 21) whose ssimilted ron, in exess for the eril orgns, is trnsloted downwrds. Following senesene sission of eril orgns t the dormnt stge, sprouting of new shoots ourred t the e of winter, oiniding with the inrese in temperture slight grdul inrese in preipittion (S1). t this stge, onentrtions were low, llowing sprouting of new shoots, similr to the report of Chope et l. (26) for sprouting uls of. ep. The higher 1-FFT tivity my hve promoted redution of frutn hin sizes, thus filitting hydrolysis y 1-FEH to supply the energy required for sprouting t S1 y the moiliztion of fruto-oligoshrides (r ¼ 79, P < 5). The inrese in the proportion of hexoses surose, ssoited with the inrese in 1-FEH tivity expression, onfirm the proximl region s the min site of frutn hydrolysis during sprouting s previously reported for this speies (seg Crvlho, 24; Portes Crvlho, 26; seg et l., 211). s sprouting shoot growth progressed (S2), the expnsion of photosynthetilly tive tissues ws ompnied y high I ontent. The low reduing sugr ontent oserved in the rhizophores in the initil phses of sprouting (S1, S2) iites fst trnslotion towrds the growing eril orgns. When the eril orgns re fully estlished (S3), the need for ron supply dereses umultion of reduing sugrs is deteted, suggesting the trnsition of rhizophores from soure to sink orgns. This sink funtion is in ordne with the high I ontent in the rhizophores, proly ting in ell

9 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Pge 9 of 13 1-FEH tivity (µg frutose mg 1 protein h 1 ) C * C * Proximl Distl * 1-FEH reltive expression (log 2 ) Proximl Distl 75 * 5 1-SST tivity (µg 1-kestose mg 1 protein h 1 ) 1-FFT tivity (µg nystose mg 1 protein h 1 ) C * expnsion neessry for reserve umultion, s shown for tuers of Helinthus tuerosus (Shuert Feuerle, 1997). The inrese in frutn synthesis ws lso deteted in llus of Viguier disolor (Ity et l., 25) in plnts of. tequiln (rreto et l., 21) supplied with uxin in vitro, giving ED S1 S2 S3 V1 V2 D Phenologil phses FIG FEH (), 1-SST () 1-FFT (C) tivities in the proximl distl rhizophore segments (see key) of plnts of Chrysolen oovt t different phenologil phses. Vlues re the mens 6 s.e. (n ¼ 3). Upper se letters iite omprison of proximl segments lower se letters iite omprison of distl segments during the experimentl period (P < 5). n sterisk (*) denotes omprison of the segments (proximl distl) within eh hrvest dte (P < 5). 1-SST reltive expression (log 2 ) 5 1 S1 S3 V1 D Phenologil phses FIG. 7. Reltive expression levels (log 2 )of1-feh () 1-SST () s ssessed y qrt-pcr nlysis in the proximl distl rhizophore segments (see key) of plnts of Chrysolen oovt t different phenologil phses (ompred with the vegettive phse). Vlues re mens (6s.e.) of two iologil replites with three tehnil replites. support to the hypothesis of reltionship etween this growth regultor frutn metolism. Speifi primers for reltive gene expression nlyses were designed on the sequene of 1-FEH from C. oovt for whih the tivity of inulin hydrolysis ws onfirmed (seg et l., 28). In the present study, higher trnsript umultion deteted in rhizophores during nturl sprouting in the field is onsistent with the inrese in 1-FEH tivity, suggesting trnsriptionl regultion of 1-FEH, s previously oserved for iued sprouting oitions (seg et l., 211). The inrese in surose reduing sugrs t the e of sprouting iued the redution of 1-FEH tivity t the eginning of the vegettive phse (V1), y feedk regultion. This my hve triggered frutn iosynthesis (Pollok Cirns, 1991), through the downwrd trnslotion of photossimiltes from the estlished eril orgns with the susequent inrese of sink pity of the rhizophores 1-SST tivity expression, orrelted with preipittion (P 1-SST ¼ 87, P < 5). ordingly, when supplied to plnts of C. oovt ultivted in vitro, surose, gluose frutose inhiited 1-FEH promoted 1-SST gene expression (F. Trevisn et l., Instituto de otâni, São Pulo, rzil, pers. omm.)

10 Pge 1 of 13 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt xis 2 (22 7 %) S2 P S3 D S3 P S2 D [I] O T [] O H2O O RS H2 Rz 1-FEH V2 P V1 P V2 D S1 P xis 1 (32 5 %) [I] Rz WP Rz [] Rz V1 D 1-SST P Oligo 1-FFT ED P D P S1 D D D ED D FIG. 8. PC i-plot of Chrysolen oovt t different phenologil phses environmentl eogenous prmeters. Wter ontent in eril orgns (H2O O) rhizophore (H2O Rz), wter potentil in rhizophore (WP Rz), temperture (T) preipittion (P) verge, ontents in eril orgns ([] O) rhizophore ([] Rz), I ontents in eril orgns ([I] O) rhizophore ([I] Rz), fruto-oligoshride (Oligo) reduing sugr (RS) ontents, 1-FEH, 1-SST 1-FFT tivities. revitions for phenologil phses re followed y P for proximl, D for distl rhizophore segments. Opposing tivities expression profiles of 1-SST 1- FEH (r ¼ 9, P < 5) were deteted, providing evidene tht plnts growing nturlly in the Cerrdo present similr ehviour in this respet to tht oserved in ultivted plnts (seg Crvlho, 24; Portes Crvlho, 26; Oliveir et l., 21), supporting the hypothesis of temporl ontrol of frutn-metolizing enzymes in the vuole, uer the regultion of surose, whih tivtes 1-SST (Wgner Wienkem, 1986) inhiits 1-FEH (Mrx et l.,1997). The mrked derese in 1-SST tivity towrds V2, lso deteted y Portes Crvlho (26) throughout the vegettive stge, is ompnied y n inrese in surose ontent higher onentrtions of. Gust et l. (25) suggested reltionship etween inresing onentrtions of sugr onentrtion to onfer old tolerne, refer to studies in whih high onentrtions of gluose /or surose re ssoited with high onentrtions of. In the present study, V2 ws ompnied y low temperture preipittion, with derese in wter potentil (W w ) of the rhizophore, the signl to mintin the wter sttus of the plnts uer low wter vilility my hve een provided y. Environmentl physiologil hnges oserved t this stge signlled the eginning of dormny(d),mrked y n inrese in 1-FEH tivity, higher fruto-oligo:fruto-polyshride rtio wter ontent in rhizophores, ll of these onverging to osmoregultion, drought old tolerne, energy supply. Temperture preipittion vlues mesured throughout the yer mthed the expeted iexes for the Cerrdo iome, espeilly for the studied re, with low preipittion during winter, refleted in low soil moisture high temperture rnge (Vuono et l., 1986; Coutinho, 22). However, the vrition of wter vilility during the yer ws not suffiient to promote hnges in plnt wter sttus, in ontrst to wht ws oserved in field experiments with Dtylis glomert L. Lolium perenne L. (Volire et l., 1998) in potted plnts of C. oovt (Gri et l., 211) V. disolor (Oliveir et l., 213) uer wter withholding. It is importnt to point out tht rhizophores of potted plnts dry out fster thn in nturl field oitions, due to the limited volume of soil surrouing it. Our results suggest tht in the Cerrdo, plnts of C. oovt present strtegies to mintin the wter sttus during the dry seson, similr to wht ws deteted in plnts of Gomphren mrgint Seu. in field oitions (Silv et l., 213). Reent works suggest tht uxin is lso involved in tolerne to drought old stress; however, the preise role of this phytohormone in ioti stress responses remins lrgely unknown. s reently shown y Shi et l. (214), trnsgeni lines of

11 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt Pge 11 of 13 Erly Dormny Dormny Sprouting Vegettive Erly Dormny Jun Jul ug Sep Ot Nov De Jn Fe Mr pr My Jun Jul Dormny ut. Winter Spring 1-FEH Summer utumn ED S1 S2 S3 V1 V2 D 1-SST/ 1-FFT Rhizophores I eril orgns Developed shoots 1-SST Winter Rhizophores Oligo I Rhizophores 1-FEH Reserve moiliztion eril growth Reduing sugrs/ Surose 1-SST Reduing sugrs/ Surose Surose eril orgns Rhizophores 1-FEH Energy supply Osmoregultion Cold tolerne Rhizophore expnsion Oligo Sprouting of eril rnhes FIG. 9. Digrm representing the min phenologil phses of Chrysolen oovt the onurrent physiologil iohemil hnges oserved.. thlin with higher eogenous uxin onentrtions show enhned resistne to drought, y positively modulting some -responsive genes the umultion of different metolites with n osmoti funtion suh s mino ids, orgni ids, surose other rohydrtes. The results reinfore the ross-tlk etween these two phytohormones in response to ioti stresses, previously reported y Du et l. (212). These uthors showed tht overexpressing gene involved in the mintenne of I homeostsis in rie resulted in redued onentrtions inresed resistne to old stress. The mintenne of I homeostsis in eril orgns rhizophores throughout the phenologil yle the deline of onentrtions in the trnsition from vegettive to dormnt phses, the oldest driest seson in the Cerrdo, suggest tht oth hormones my e essentil for C. oovt to ope with the limti oitions during winter. The PC onfirms wht ws pointed out previously, iiting the influene of 1-FEH in sprouting, espeilly in S1, 1-SST in vegettive periods, s well s their opposing tivities. The reltionship fou etween tempertures,, I wter ontents in eril orgns with phses in whih plnts presented developing or well-estlished eril orgns (inluding ED) ws opposite to tht in whih plnts did not hve eril orgns tempertures were lower, suh s dormny the eginning of sprouting. The ssoition etween in the rhizophore erly dormny, in spite of the high 1-SST tivity, iited the entry of plnts in dormny. The digrm presented in Fig. 9 summrizes the phenologil phses of C. oovt, its mehnisms of dpttion the strtegies to eure ioti stresses previling in the Cerrdo. Chrysolen oovt is le to up- downregulte frutn metolism in response to environmentl hnges, enling plnts to uergo unfvourle periods. Eogenous hormone onentrtions seem to e relted to regultion of frutn metolism to the trnsition etween phenophses, signlling for energy storge, reserve moiliztion umultion of oligoshrides s osmolytes. In ddition, vrition in hormonl ontents ould diretly ontriute to tolerne to ioti stresses, modulting pthwys other thn just those relted to rohydrtes, not essed in this study. Temperture photoperiod ould e other signls for the trnsition etween phenophses; however, other environmentl ftors, suh s rdition levels reltive ir humidity, must lso e onsidered in future studies. SUPPLEMENTRY DT Supplementry dt re ville online t onsist of the following. Figure S1: imgeof C. oovt, highlighting the proximl distl regions of rhizophores. Figure S2: soil wter retention urves. Tle S1: list of primers used for qrt-pcr. CKNOWLEDGEMENTS We thnk the Instituto gronômio de Cmpins (Núleo de Irrigção e Drengem) for performing the wter soil retention

12 Pge 12 of 13 Rigui et l. Eogenous hormones frutn metolism in Chrysolen oovt nlysis, E.. Silv C. Ferrgut for helping with nlyses of wter sttus PC, respetively, J. del Giudie for support during field work t the iologil Reserve, F. Trevisn for tehnil support in qrt-pcr expression nlysis, R. C. L. Figueiredo-Rieiro for sientifi dvie. This work ws supported y Conselho Nionl de Desenvolvimento Científio e Tenológio-CNPq (grnt no. 4785/211-3) Plno Nionl de poio o Desenvolvimento d otâni/coordenção de perfeiçomento de Pessol de Nível Superior PND/CPES (grnt no. 454/21)..P.R. ws finnilly supported y PND/CPES, M..M.C. is CNPq Reserh Fellow. LITERTURE CITED seg F, Crvlho MM. 24. Frutn metolising enzymes in rhizophores of Vernoni here upon exision of eril orgns. Plnt Physiology iohemistry 42: seg F, Nsimento JRO, Shroeven L, Vn den Ee W, Crvlho MM. 28. Cloning, hrteriztion funtionl nlysis of 1-FEH DN from Vernoni here (Vell.) Rusy. Plnt Cell Physiology 49: seg F, Nsimento JR, Crvlho MM Inresed expression of frutn 1-exohydrolse in rhizophores of Vernoni here during sprouting exposure to low temperture. Journl of Plnt Physiology 168: rreto R, Nieto-Sotelo J, Css GI. 21. Influene of plnt growth regultors wter stress on rmet iution, rosette engrossment, frutn umultion in gve tequiln Weer vr. zul. Plnt Cell, Tissue Orgn Culture 13: tlh M, Mntovni W. 2. Reprodutive phonologil ptterns of Cerrdo plnt speies t the pé-de-gignte reserve (Snt Rit do Pss Qutro, SP, rzil): omprison etween the hereous woody flors. Revist rsileir de iologi 6: lke GR ulk density. In: C lk, DD Evns, JL White, LE Ensminger, FE Clrk, eds. Methods of soil nlysis. Mdison, WI: merin Soiety of gronomy, rdford MM rpid sensitive method for quntifition of mirogrm quntities of proteins utilizing the priniple of protein dye iing. nlytil iohemistry 72: rvo L, Zúñig GE, lerdi M, Coruer LJ The role of in freezing tolerne old limtion in rley. Physiologi Plntrum 13: ustin S, enes V, Grson J, et l. 29. The MIQE guidelines: minimum informtion for pulition of quntittive rel-time PCR experiments. Clinil Chemistry 55: Crvlho MM, Dietrih SMC Vrition in frutn ontent from uergrou orgns of Vernoni here (Vell.) Rusy in different phenologil phses. New Phytologist 123: Crvlho MM, seg F, Figueiredo-Rieiro, RCL. 27. Frutns in steree from rzilin Cerrdo. In: S Norio, Noureddine, O, Shuihi, eds. Reent dvnes in frutooligoshrides reserh. Kerl, Ii: Reserh Signpost, Chope G, Terry L, White PJ. 26. Effet of ontrolled tmosphere storge on sisi id onentrtion other iohemil ttriutes of onion uls. Posthrvest iology Tehnology 39: Coutinho LM. 22. Eugen Wrming e o Cerrdo rsileiro: um séulo depois..l. Klein, org. São Pulo: UNESP/Imprens ofiil do Estdo de São Pulo. Cruz CR, Lirdi PL, Crvlho L, Roh GC. 25. lnço de águ no volume de solo explordo pelo sistem rdiulr de um plnt de itrus. Revist rsileir de Ciêni do Solo 29:1 1. 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Mrx SP, Nöserger J, Frehner M Sesonl vrition of frutn--frutosidse (FEH) tivity hrteriztion of -(2,1)-linkge speifi FEH from tuers of Jeruslem rtihoke (Helinthus tuerosus). New Phytologist 135: Mdm SM, rodri TJ Hormonl dynmis ontriutes to divergene in sesonl stomtl ehvior in monsoonl plnt ommunity. Plnt, Cell Environment 38: Mihiels, Vn Lere, Vn den Ee W, Tuker M. 24. Expression nlysis of hiory frutn 1-exohydrolse gene revels omplex regultion y old. Journl of Experimentl otny 55: Oliveir VF, Zidn LP, rg MR, idr MPM, Crvlho MM. 21. Elevted CO 2 tmosphere promotes plnt growth inulin prodution in the errdo speies Vernoni here. Funtionl Plnt iology 37: Oliveir VF, Silv E, Zidn LP, Crvlho MM Effets of elevted CO 2 onentrtion wter defiit on frutn metolism in Viguier disolor ker. Plnt iology 15: Pfffl MW. 21. new mthemtil model for reltive quntifition in reltime RT-PCR. 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