Adaptive strategies of Parietaria diffusa (M.&K.) to calcareous habitat with limited iron availability*pce_

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1 s_s_nner Plnt, Cell nd Environment (2012) 35, doi: /j x Adptive strtegies of Prietri diffus (M.&K.) to clcreous hitt with limited iron vilility*pce_ SILVIA DONNINI, PATRIZIA DE NISI, DAMIANO GABOTTI, LILIANA TATO & GRAZIANO ZOCCHI Diprtimento di Produzione Vegetle, Università degli Studi di Milno, vi Celori 2, Miln, Itly ABSTRACT The study of ntive plnts growing in hostile environments is useful to understnd how these species respond to stress conditions. Prietri diffus (M.&K.) is le to survive in highly clcreous soils nd extreme environments, such s house wlls, without displying ny chlorotic symptoms. Here, we hve investigted the existence of Strtegy I complementry/lterntive mechnism(s) involved in Fe soluiliztion nd uptke nd responsile for Prietri s extrordinry efficiency. After ssessing the specific trits involved in clcicole-ehviour in the field, we hve grown plnts in conditions of Fe deficiency, either direct (-Fe) or induced y the presence of icronte (+FeBic). Then, the growth performnce, physiologicl nd iochemicl responses of the plnts were investigted. The study shows tht in Prietri +FeBic, the clssicl responses of Strtegy I plnts re ctivted to lower extent thn in -Fe. In ddition, there is greter production of phenolics nd orgnic cids tht re oth exuded nd ccumulted in the roots, which in turn show structures similr to proteoid-like roots. We suggest tht in the presence of this constrint, Prietri undergoes some metolic rerrngements tht involve PEP-consuming rections nd n enhncement of the shikimte pthwy. Key-words: icronte; clcreous soils; Fe deficiency; orgnic cids; phenolics. INTRODUCTION In n ecosystem, the coloniztion y specific plnt species ffects iodiversity. However, there re few studies out the physiologicl properties of species tht determine their success during ecologicl competition nd therefore their environmentl distriution. The chemico-physicl chrcteristics of the soil re often importnt fctors tht strongly control plnt distriution nd growth. It is well known tht the presence of icronte nd high ph in the soil cuse diminished iovilility of some nutrients, minly Fe, inducing in plnts common nutritionl disorder nmed chlorosis (Lindsy 1984). Given Correspondence: S. Donnini, Fx: ; e-mil: silvi. donnini@unimi.it *This work ws supported y grnts from MIUR nd from the Università degli Studi di Milno. tht more thn 30% of the soils in the world re clcreous, it is quite common tht crops must fce this prolem (Chen & Brk 1982). Plnts show different susceptiility to soil fctors inducing Fe deficiency, so tht different degree of stress nd gret vriility exist in response to Fe deprivtion. A vriility in Fe cquisition cpcity hs een found in spontneous vegettion (Nelson 1992), nd ntive plnts cn e ssigned to Fe-efficient nd Fe-inefficient clsses on the sis of the presence nd diffusion in pedologicl environments inducing low Fe vilility. In prticulr, the so-clled clcicole species seem to strongly colonize clcreous hitts etter thn other species (i.e. clcifuge plnts), demonstrting etter efficiency in otining Fe under these unfvourle environments. In fct, it hs een demonstrted tht clcicole ntive dicotyledonous plnts re more Fe efficient thn clcifuge ones, showing higher Fe 3+ -chelte reductse (FC-R) ctivity, response ssigned to the Strtegy I (Schmidt & Brtles 1998). Strtegy I is complex Fe uptke mechnism developed y ll plnts, with the only exclusion of the Pocee, which elongs to Strtegy II (Römheld & Mrschner 1986; Morrissey & Guerinot 2009; Adí et l. 2011). This Fe uptke mechnism is sed on the reduction of externl Fe 3+ to Fe 2+ through the induction of Fe 3+ -chelte reductse enzyme loclized t the plsm memrne of the rhizoderml cells (Roinson et l. 1999; Wters, Blevins & Eide 2002; Li, Cheng & Ling 2004). Once reduced, the Fe 2+ ion is trnsported inside the roots y crrier (IRTs) elonging to the ZIP fmily of trnsporters (Guerinot 2000; Henriques et l. 2002; Vrotto et l. 2002; Vert et l. 2002). The cpcity to grow in Fe-poor environments is not restricted only to the induction of the two previously mentioned mechnisms, ut other ctivities cn e induced during Fe deficiency stress. Among these, severl iochemicl nd morphologicl dpttions occur in Strtegy I plnts. One of the most importnt is the enhnced cpcity to decrese the rhizospheric ph through the ctivtion of plsm memrne-loclized P-type H + -ATPse (Zocchi & Cocucci 1990; Plmgren 2001; Snti & Schmidt 2009). Proton extrusion is useful oth to increse Fe vilility y incresing the soluility of Fe compounds (Dell Orto et l. 2000), s well s to set up fvourle trnsmemrne electricl potentil (negtive inside) for ction uptke. However, the induction of the reduction processes nd the enhnced extrusion of protons under Fe deficiency need n incresed rte of NAD(P)H nd ATP regenertion, which is chieved 2012 Blckwell Pulishing Ltd. 1171

2 1172 S. Donnini et l. through the ccelertion of metolism, in prticulr glycolysis. Among the metolic ctivities which re incresed under Fe deficiency, phosphoenolpyruvte croxylse (PEPC) seems to ply pivotl role (Zocchi 2006 nd references therein). In fct, the enzyme is ctive in severl nplerotic rections to replenish tricroxylic cid cycle intermedites, to provide cron skeletons to sustin the synthesis of mino cids nd for the ph-stt mechnism (De Nisi & Zocchi 2000; López-Millán et l. 2000; Zocchi 2006). In some Strtegy I species lso, the relese of orgnic compounds such s phenolics, flvins, sugrs nd orgnic cids could help in the soluiliztion of Fe-contining compounds (Welkie 2000; Curie & Brit 2003; Jin et l. 2007; López-Millán et l. 2009). Considering the reducing nd complexing proprieties of phenolic compounds, it is widely ccepted tht the mechnism y which they cn regulte Fe moility in the rhizosphere might ply n importnt role (Jin et l. 2007; Tomsi et l. 2008; Cesco et l. 2010). Recently, few studies hve explored the role of secondry metolic pthwys in plnt response to Fe deficiency. An exmple is the shikimte pthwy, which is responsile for the synthesis of phenolic compounds. Some of the key enzymes ctlyzing this pthwy include shikimte dehydrogense (SDH), shikimte kinse (SK) nd phenyllnine mmoni lyse (PAL) (M sehli et l. 2009; Ln et l. 2011). From morphologicl point of view, the ppernce of swollen tips, secondry lterl roots nd root hirs increse the surfce of contct etween roots nd soil, fvouring the serch nd cquisition of nutrients (Lndserg 1996; Schmidt & Brtels 1996). Induction of these different responses, in ddition to reduction nd trnsport processes, would increse plnt efficiency in solving the prolem of Fe deficiency. The more plnt is le to diversify its response, the more efficient will e the result. In lkline nd clcreous environments, Fe soluility nd vilility re drsticlly reduced long with other nutrients (for instnce P) (Guerinot & Yi 1994; von Wndruszk 2006). This mens tht in these conditions, reltively low mount of solule nutrients might e ville for plnt uptke nd therefore for use in vrious physiologicl functions, ffecting photosynthesis, growth, competition nd survivl of the species. However, hevy clcreous sites re colonized y plnts with morpho-physiologicl chrcteristics of resistnce to these prticulr conditions. Prietri diffus, spontneous species lso nmed pellitory of the wll, is dicot elonging to the Urticcee fmily, widespred in the Mediterrnen re, United Sttes nd Austrli. It is le to grow on wlls, deris nd sustrtes with very high cronte concentrtion without showing ny Fe-deficiency symptom (Fig. 1). For this reson, Prietri could represent model plnt to understnd the ecophysiologicl trits determining the clcicole ehviour of plnts nd the mechnisms of resistnce developed y them to fce with these dverse conditions, prticulrly concerning Fe deficiency. In previous work (Dell Orto et l. 2003), we hve chrcterized in prt the iochemicl nd physiologicl Figure 1. Spontneously growing Prietri diffus (M.&K.). responses of Prietri under Fe-deficiency conditions, typicl of clcreous hitt, in order to clrify whether it ehves s Strtegy I plnt. In the present study, some secondry responses to Fe deficiency were further investigted, with the im to determine the existence of lterntive or complementry mechnism(s) to the typicl Strtegy I, which my e responsile for Prietri s extrordinry efficiency in Fe cquisition. In prticulr, to identify the specific trits of tolernce involved in clcicole ehviour, n nlysis on Prietri plnts collected directly from wlls with different ph vlues hs een crried out. Then, Prietri plnts hve een grown in hydroponics under Fe deficiency, either direct or induced y the presence of icronte, nd the morphologicl, physiologicl nd the iochemicl trits of the plnts hve een investigted. MATERIALS AND METHODS Plnt mteril Field smpling P. diffus (M.&K.) plnts were smpled in three different sites ner the University of Miln (Itly). Smpling res were chosen priori sed on the presence of Prietri s the unique species. Smples were collected y reking the wlls nd the sustrtes surrounding roots were lso collected nd soluilized in distilled wter in order to determine the ph (ph-meter PHM 240 ph/ion Meter). Hydroponic culture Cuttings of Prietri were otined from mother plnt nd put in n erted hlf-strength nutrient solution for 1 week to rdicte. Once rooted, plnts were trnsferred to 10 L plstic pots (40 plnts/pot) contining (1) full nutrient solution plus 100 mm Fe(III)-EDTA (+Fe), ph 6.2; (2) full nutrient solution without Fe (-Fe), ph 6.2; (3) full nutrient solution with 100 mm Fe(III)-EDTA, 15 mm NHCO 3 nd 0.5gL -1 CCO 3 (+FeBic), which rought the ph to 8.3. An 2012 Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

3 Adptive strtegy to limited iron vilility 1173 ddition of 0.5 g L -1 CCO 3 is useful to keep the icronte concentrtion constnt to pproximtely 15 mm throughout the experiment. The composition of the full strength solution ws s reported in Donnini et l. (2010). Plnts were grown for 9 d under different tretments in growth chmer under 16/8 h light/drk regime, 27/21 C, 65 75% reltive humidity nd with PPDF of 200 mmol m -2 s -1. Collection of root exudtes Root exudtes were collected ccording to Gries et l. (1995). Five plnts from ech tretment were trnsferred to 250 ml of distilled wter, with the ph djusted to 6.2, contining 10 mg L -1 of Micropur (Ktdyn, Minnepolis, MN, USA) to prevent decomposition of orgnic mtter y microorgnisms. Root exudtes were collected over 24 h period in growth chmer under continuous ertion nd freeze dried. The lyophilized mteril ws resuspended in distilled wter nd filtered through 0.45 mm Millipore Millex-HN (Billeric, MA, USA); oth phenolics nd orgnic cid concentrtions were then determined. For ech tretment, five replictes were performed. Determintion of phenolic compounds Determintion of phenolics concentrtion in the roots ws performed using two different extrction solutions: distilled wter or methnol. Smpled roots were homogenized in 1 volume (w/v) of extrction solution nd the homogente centrifuged t g for 10 min. Extrcts were filtered through 0.45 mm Millipore Millex-HN. The concentrtion of phenolics in oth roots nd exudtes ws determined spectrophotometriclly t 750 nm with the Folin Cioclteu regent ccording to the method of Swin & Hillis (1959), using gllic cid s stndrd. For ech tretment, five replictes were performed. Orgnic cids ssy For the quntittive determintion of orgnic cids, roots were collected, crefully rinsed in distilled wter nd homogenized in the presence of 5 ml of 10% (v/v) perchloric cid nd centrifuged t g for 15 min. The ph ws rought to 7.5 with 0.5 M K 2CO 3 to neutrlize the cidity nd to precipitte the perchlorte. Extrcts were clrified y centrifugtion t g for 15 min. Citric nd mlic cid concentrtion in oth roots nd exudtes ws determined enzymticlly, using specific kits from Boehringer Mnnheim (Mnnheim, Germny) nd ccording to the mnufcturer s instructions. The recovery of oth orgnic cids ws more thn 90% s determined y the use of n internl stndrd (Rotti, De Nisi & Zocchi 1995). For ech tretment, five replictes were performed. Biomss mesurement nd lef chlorophyll determintion Growth of shoots nd roots ws clculted s the difference etween the end (9 d) nd the eginning of tretments Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35, Fresh weight (FW) nd dry weight (DW) were determined t the end of tretments. Three independent experiments in triplicte (n = 9) were performed. Chlorophyll concentrtion ws determined ccording to Lichtenthler (1987). For ech tretment, five replictes were performed. Determintion of Fe nd P For the determintion of poplstic Fe, plnts from ech tretment (n = 5) were trnsferred to eker with 0.5 mm CSO 4 under vigorous ertion. After 10 to 15 min, plnts were plced with their root system in 40 ml tues with 21 ml of 10 mm MES, 0.5 mm C(NO 3) 2, 1.5 mm 2,2 - ipyridyl (ph 5.5) t 25 C. Tues were covered with cotton plug nd N 2 ws uled through the solution. After 5 min, 1 ml of 250 mm N 2S 2O 4 ws dded from syringe. The A 520 of the solution [A 520 of 1 mm Fe(ipyridyl) 3 = 8.650] ws determined on 2 ml liquots s reported y Bienfit, vn den Briel & Meslnd-Mul (1985). After removl of poplstic Fe, the mount of Fe nd P ccumulted in the sme plnt ws determined. For this, roots nd leves were collected seprtely, crefully rinsed with distilled wter nd oven dried t 55 C, ground to powder nd minerlized in 1 M HNO 3. Finl volumes were djusted to 1 ml y 0.1 M HNO 3. Fe ws determined y ICP-OES (Sequentil ICP-OES AX Lierty, Vrin) nd P y ICP-MS (Vrin 820-MS). Fe reduction nd cidifiction in vivo Visuliztion nd locliztion of Fe reduction nd cidifiction ctivities of the roots were performed using the gr technique s reported y Donnini et l. (2009). Root segments were extensively wshed until the ph in the solution ws stle t ph 6.1 nd emedded in 0.75% (w/v) gr contining 100 mm Fe(III)-EDTA nd 100 mm thophennthroline disulfonte (BPDS) for reduction ctivity or 0.006% romocresol purple (ph indictor) for cidifiction ctivity. The experiment ws repeted three times with the sme results. Fe reduction y phenolics exuded nd ccumulted in the roots The phenolics ility to reduce Fe(III)-EDTA ws mesured spectrophotometriclly using the BPDS regent (Chney, Brown & Tiffin 1972). Phenolics ( mg) prepred ccording to the method descried previously were incuted for 120 min in 1 ml of solution contining 100 mm Fe(III)-EDTA nd 100 mm BPDS in the drk t 26 C under shking. The sornce t 535 nm ws determined s reported y Donnini et l. (2009). The experiment ws repeted three times. Preprtion of plsm memrne vesicles Roots of 9-dy-old plnts were excised, rinsed in distilled wter nd homogenized in mortr t 2 4 C in uffer

4 1174 S. Donnini et l. contining 50 mm MOPS-BTP ph 7.5, 330 mm sucrose, 5mm EDTA, 1 mg ml -1 ovine serum lumin (BSA), 5mmdithiothritol (DTT), 2 g ml -1 PMSF, 10% (w/v) diethyldithiocrmic cid sodium slt (DIECA). After filtrtion through four lyers of guze, the homogente ws centrifuged t g for 15 min nd the superntnt ws centrifuged t g for 30 min to otin microsoml frction. The plsm memrne-enriched frction ws prepred using the two-phse prtitioning procedure s reported y Lrsson, Sommrin & Widell (1994). The enrichment in plsm memrne ws determined ssying vndte-sensitive H + -ATPse (EC ) (PM), nitrte-sensitive H + -ATPse (EC ) (tonoplst) nd zide-sensitive H + -ATPse (EC ) (mitochondri), respectively, ccording to Rotti & Zocchi (1994). H + -ATPse ssy H + -ATPse ctivity in plsm memrne preprtions ws determined in medium with the following composition: 250 mm sucrose, 50 mm KCl, 25 mm MOPS-BTP ph 6.5, 1mm ATP, 0.25 mm NADH, 1 mm PEP, 15 mgml -1 lctte dehydrogense (EC ), 30 mgml -1 pyruvte kinse (EC ), 0.015% (w/v) Lurol nd mg plsm memrne protein (omitted in lnks). The rection ws strted y the ddition of 1 mm MgSO 4. NADH oxidtion ws followed t 340 nm. Three independent experiments in triplicte (n = 9) were performed. FC-R ssy The NADH-dependent Fe(III)-reductse ctivity of plsm memrne preprtions isolted from roots ws determined t 26 C in medium contining 250 mm sucrose, 15 mm Mops-BTP ph 7, 0.25 mm K 3Fe(CN) 6, 0.25 mm NADH nd 0.01% (w/v) Lurol, nd the rection ws strted y the ddition of mg of plsm memrne protein (omitted in lnks). The sornce chnge t 340 nm ws monitored. Three independent experiments in triplicte (n = 9) were performed. Solule protein extrction nd PEPC ssy Roots of 9-dy-old plnts grown under the different tretments were hrvested, rinsed nd homogenized in uffer (1 ml g -1 FW) contining 50 mm Tris-HCl ph 7.5, 10 mm MgCl 2, 10% (v/v) glycerol, 1 mm EDTA, 14 mm -mercptoethnol, 1 mm PMSF nd 10 mgml -1 leupeptin. The homogente ws filtered through four lyers of guze nd centrifuged t g for 15 min nd the superntnt ws gin centrifuged t g for 30 min. PEPC (EC ) ws determined s in De Nisi & Zocchi (2000).The rection ws strted y the ddition of ml of solule frction (omitted in lnks) nd the sornce chnge t 340 nm ws monitored. Three independent experiments in triplicte (n = 9) were performed. Western lot nlysis of PEPC nd H + -ATPse Solule nd plsm memrne proteins (15 mg) were loded on discontinuous sodium dodecyl sulphte-polycrylmide gel electrophoresis (SDS-PAGE) (3.75% (w/v) crylmide stcking gel nd 8% (w/v) crylmide seprting gel). After SDS-PAGE, Western lot nlyses were performed s reported in De Nisi & Zocchi (2000) for PEPC nd in Dell Orto et l. (2000) for the H + -ATPse, respectively. Two different ntiser were used; one rised ginst the centrl domin of PM H + -ATPse of Aridopsis thlin ( kind gift from Dr R. Serrno) nd second one rised ginst PEPC isoform of sorghum ( kind gift from Dr J.Vidl).The experiment ws repeted three times with the sme result. Shikimte pthwy enzyme extrction nd ssy For 3-deoxy-D-rino-heptulosonte-7-phosphte synthse (DAHPS, EC ), 0.2 g fresh roots were homogenized in 100 mm potssium-phosphte uffer (ph 8.0) contining 1.4 mm -mercptoethnol. The homogente ws centrifuged t g for 15 min t 4 C nd DAHPS ctivity ws ssyed on solule frction s reported y Sánchez-Rodríguez et l. (2011). SDH nd SK extrction ws performed ccording to Díz, Brceló & Merino (1997). SDH (EC ) ws ssyed t 25 C y monitoring the reduction of NADP + t 340 nm ccording to Chudhuri & Coggins (1985). The ssy mixture contined ml of solule frction (omitted in lnks) in 100 mm N 2CO 3 ph 10.6, 4 mm shikimic cid nd 2 mm NADP +. SK (EC ) ws ssyed t 25 C y coupling the relese of ADP to the oxidtion of NADH using pyruvte kinse (EC ) nd lctte dehydrogense (EC ) s coupling enzymes ccording to Krell et l. (2001). Shikimte-dependent oxidtion of NADH ws monitored t 340 nm. The ssy mixture contined ml of solule frction (omitted in lnks) in 50 mm triethnolmine hydrochloride/koh uffer t ph 7.0, 50 mm KCl, 5 mm MgCl 2, 1.6 mm shikimic cid, 5 mm ATP, 1mm PEP, 0.1 mm NADH, 30 mgml -1 pyruvte kinse nd 15 mgml -1 lctte dehydrogense. The enzyme 5-enol-pyruvyl-shikimte-3-phosphte (EPSP) synthse (EC ) ws extrcted nd ssyed s reported y Forlni, Nielsen & Rcchi (1992). EPSP synthse ctivity ws mesured in medium contining 100 mm Hepes NOH ph 7.4, 1 mm shikimte-3-phosphte (S3P), 1 mm PEP, 0.5 mm mmonium heptmolydte nd 60 ml of enzyme. After incution, the rection ws stopped y ddition of solution contining mlchite green nd smples were red t 660 nm ginst lnks in which S3P hd een omitted. Phenyllnine mmoni-lyse (PAL) (EC ) ws otined y homogenizing 0.20 g fresh tissue in 15 ml of n extrction medium contining 20 mm -mercptoethnol, 0.1 M sodium orte uffer ph 8.8 nd 5% (w/v) PVPP. After filtrtion through four lyers of guze, the 2012 Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

5 Adptive strtegy to limited iron vilility 1175 homogente ws centrifuged t g for 20 min, 4 C. The enzyme ctivity ws determined y dding 1 ml of the protein extrct (omitted in lnks) to rection medium ccording to Chill & Mc Com (1992). All the enzymes were ssyed y three independent experiments in triplicte (n = 9). Protein determintion Protein content ws determined y the Brdford (1976) procedure using BSA s stndrd. Oxygen consumption Apicl root segments (2 cm) were excised under wter t room temperture from plnts illuminted for severl hours. Root O 2 consumption rtes were mesured from the decrese in O 2 concentrtion in n queous phse with Clrk-type O 2 electrode (YS1 Anlyticl control) t 25 C. Clirtion ws mde from the difference in signl etween erted wter nd N-dithionite sturted wter. The effects of KCN (2 mm) nd slicylhydroxmic cid (SHAM) (2 mm) were determined s reported y Vigni, Mffi & Zocchi (2009). Five replictes for ech tretment were performed. Sttistics All sttisticl nlyses were conducted with SigmStt 3.1 (Systt Softwre, Chicgo, IL, USA). Mens were compred y t-test t P < 0.05 in ll cses. RESULTS Orgnic compounds in roots nd exudtes Tle 1 shows the results for Prietri roots smpled oth in the field nd in hydroponic culture. Concerning the first ones, we oserved correltion etween the increse in ph of the sustrtes surrounding roots nd phenolics ccumultion in the root. A similr trend ws lso oserved for mlic nd citric cid concentrtions, which incresed in the roots when the ph of the growing medium incresed. In Prietri plnts grown in hydroponic culture, the sence of Fe induced n ccumultion of phenolics in the roots (out 3- nd 4-fold when extrcted in wter nd methnol, respectively). Furthermore, in the presence of icronte, n increse in phenolics concentrtions occurred in the roots (7- nd 22-fold when extrcted in wter nd methnol, respectively). Both mlic nd citric cid concentrtions were found to increse (8% nd 5-fold, respectively) s Fe in the hydroponic culture ws removed. However, when icronte ws dded to the +Fe solution, the concentrtions of orgnic cids were significntly higher (3.3- nd 8.3-fold increses for mlic nd citric cid, respectively). Concerning phenolics concentrtions in the exudtes, n increse in -Fe plnts (+18%) nd in plnts grown in the presence of icronte (+61%) ws detected. The mlic cid concentrtion in exudtes of plnt exposed to -Fe condition ws significntly higher thn in plnts grown in the presence of icronte, wheres for citric cid the mjor increse ws found in the +FeBic tretment (Tle 1). With respect to the reltive controls, the mlic cid concentrtion incresed y 4.6-fold nd 3-fold in -Fe nd +FeBic tretments respectively, wheres citric cid concentrtion incresed y out 18-fold nd 33-fold in -Fe nd +FeBic, respectively. Biomss mesurement Plnt growth ws influenced y the stress conditions nd significnt decrese in shoot length ws found oth in the sence of Fe (-31%) nd in the presence of icronte (-55%). Root length lso decresed when icronte ws dded to the medium (-74%), while the -Fe tretment did not cuse significnt decreses in this prmeter (Fig. 2). Tle 1. Wter nd methnol solule phenolics, mlic cid nd citric cid content in Prietri roots nd exudtes Smple Wter-solule phenolics (mg g -1 FW) Methnol-solule phenolics (mg g -1 FW) Mlic cid (mg g -1 FW) Citric cid (mg g -1 FW) Prietri in the field ph c c ph ph Prietri in hydroponic +Fe (ph 6.2) c c c c -Fe (ph 6.2) FeBic (ph 8.3) Exudtes in hydroponic +Fe (ph 6.2) c c c -Fe (ph 6.2) FeBic (ph 8.3) Plnts were collected either from the field or from the hydroponic culture. Growth conditions re reported s in Mterils nd Methods section. Dt re mens SD (n = 5). In the cse of significnt differences (P < 0.05), vlues re mrked with different letters. FW, fresh weight Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

6 1176 S. Donnini et l. () cm () g (c) g c c c the presence of icronte (-17 nd -26%, respectively), no significnt difference ws detected t the lef level. Root Fe reduction nd medium cidifiction in vivo In order to otin preliminry overview of the induction of Fe deficiency responses, the reduction nd the cidifiction cpcities were crried out in vivo y emedding the roots in gr (Fig. 4). The reduction ctivity ws detected y using BPDS, which forms stle red complex only with Fe 2+ (Römheld, Müller & Mrschner 1984), indicting tht reduction of Fe 3+ hs occurred. Figure 4 shows tht plnts grown in presence of Fe did not develop ny colour, nd conversely in -Fe-treted plnts high reduction ctivity ws present, minly loclized in the picl nd su-picl zones (Fig. 4). Prietri plnts grown with icronte showed the formtion of red colour less intense thn -Fe-treted plnts (Fig. 4c). However, in this tretment, the reduction ctivity ppered to e more diffuse long the root length. Medium cidifiction ws determined in vivo y the chnge in the colour of the ph indictor romocresol The FW of the shoot ws reduced when plnts were exposed to Fe deficiency (-26%) nd icronte (-40%). No significnt differences were found in root FW (Fig. 2). The plnt DW showed the sme trends (Fig. 2c). Lef chlorophyll determintion Figure 3 shows the chlorophyll concentrtion expressed on n FW sis. The totl lck of Fe resulted in lrger decrese in lef chlorophyll concentrtion with respect to the icronte supply (-34 nd -20%, respectively). Fe nd P determintion +Fe -Fe +FeBic Figure 2. Shoot nd root length (), fresh weight () nd dry weight (c). The cler prt of the histogrm corresponds to prmeters determined on the shoots; the drk re indictes the prmeters determined on the roots. Three independent experiments in triplicte (n = 9) were performed. In the cse of significnt differences (P < 0.05), vlues re mrked with different letters. Tle 2 reports the mount of poplstic Fe in the roots from different growth conditions. The Fe trpped in the free spce of roots grown in the presence of icronte incresed 4.9-fold, while in -Fe decrese (-19%) ws found. In the sme plnts, decrese in Fe concentrtion ws detected only in the tissues of -Fe roots, while t the shoot level the decrese ws oserved in oth -Fe nd +FeBic tretments (-70 nd -53%, respectively). Concerning P concentrtion, different trend ws oserved: while in the roots slight decrese ws found in sence of Fe nd in Chlorophyll content (mg g -1 FW) c +Fe -Fe +FeBic Figure 3. Chnge in the lef chlorophyll concentrtion (mg g -1 FW) in plnts grown for 9 d in control condition (+Fe), Fe deficiency (-Fe) nd in the presence of icronte (+FeBic). Dt re the mens SD (n = 5). In the cse of significnt differences (P < 0.05), vlues re mrked with different letters. FW, fresh weight Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

7 Adptive strtegy to limited iron vilility 1177 Fe Shoot Root simplst Root poplst Shoot Root +Fe Fe c c FeBic P Tle 2. Fe nd P concentrtion (mmol g -1 DW) in leves nd roots of plnts grown for 9 d in control condition (+Fe), iron deficiency (-Fe) nd in the presence of icronte (+FeBic) Dt re mens SD (n = 5). In the cse of significnt differences (P < 0.05), vlues re mrked with different letters. DW, dry weight. purple nd it is shown in Fig. 4d f. The pictures reported in the figure were tken pproximtely 2 h fter gr emedding nd show n cidifiction ctivity only in -Fe nd +FeBic conditions (Fig. 4e,f). The difference with the locliztion of the reduction ctivity is noteworthy, s the cidifiction ppered more diffuse in the -Fe-treted roots nd more loclized in the roots of icronte-treted plnts, prticulrly in the zone where the proteoid-like roots were present. Fe reduction ility y phenolics Tle 3 shows the results otined testing the Fe reduction ility y oth exuded nd root ccumulted phenolics. In prticulr, even if n ctivity ws detected for ll the root Tle 3. Fe(III) reduction ctivity y phenolics extrcted from Prietri roots nd plnt exudtes Root extrcts Wter solule Methnol solule Plnt exudtes +Fe c -Fe FeBic Reduction is expressed s mmol Fe mg -1 phenolics h -1. Dt re mens SD (n = 3). In the cse of significnt differences (P < 0.05), vlues re mrked with different letters Figure 4. Visuliztion of Fe reduction (,,c) nd cidifiction (d,e,f) ctivities of the whole root system. Roots were emedded in 0.75% gr medium nd the reduction ws determined, fter out 2 h, s the Fe 2+ -[thophennthroline disulfonte (BPDS) 3] complex formtion (red), wheres the cidifiction ws detected s ph chnges of the indictor romocresol purple (yellow). From left: 9-dy-old root grown under control condition (,d), Fe deficiency (,e) nd +FeBic tretment (c,f). The experiment ws repeted three times with similr results Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

8 1178 S. Donnini et l. Enzymes +Fe -Fe +FeBic H + -ATPse nmol min -1 mg -1 prot c Vndte sensitive Nitrte sensitive Azide sensitive FC-R nmol min -1 mg -1 prot c PEPC nmol min -1 mg -1 prot c Tle 4. Enzymtic ctivities of typicl Strtegy I responses in root plsm memrne preprtions nd root solule extrcts from control (+Fe), Fe-deficient (-Fe) nd icronte (+FeBic)-treted plnts For the H + -ATPse, the mrker enzyme ctivities in plsm memrne-enriched frctions re reported. Dt re the mens SD (n = 9). In the cse of significnt differences (P < 0.05), vlues re mrked with different letters. PEPC, phosphoenolpyruvte croxylse; FC-R, Fe(3+)-chelte reductse. extrcts, higher vlues were found for the +FeBic tretment. Regrding the exuded phenolics, n increse in Fe reduction ility ws detected for oth -Fe (+17%) nd +FeBic (+32%) with respect to the control. Strtegy I response ctivities in plsm memrne preprtions nd root solule extrcts The ssy of mrker enzyme ctivities (vndte-sensitive, nitrte-sensitive nd zide-sensitive ATPses) crried out on plsm memrne preprtions isolted from roots of differently treted plnts indicte tht the H + -ATPse ctivity ws scrcely sensitive to nitrte nd zide (less thn 6% inhiition), while it ws lmost completely inhiited y vndte (c. 90%), indicting n enrichment in plsm memrne in the preprtions used (Tle 4). On these enriched preprtions we hve nlysed the Fe 3+ -chelte reductse nd the H + -ATPse ctivities (Tle 4). Both Fe-deficiency conditions induced n increse in FC-R ctivity in plsm memrne preprtions from roots of P. diffus; in prticulr, in -Fe tretment the enhncement ws pproximtely 2.3-fold with respect to control plnts, while in the presence of icronte the increse in FC-R ctivity ws only pproximtely +51%. The H + -ATPse ctivity showed the sme trend: oth -Fe nd +FeBic tretments showed n incresed ctivity with respect to the control, ut lower for the icronte tretment (+95 nd +45%, respectively). PEPC ctivity in root solule extrcts ws differently ffected in plnts grown under Fe sence nd icronte supply conditions. In fct, n increse (+74%) in PEPC ctivity ws oserved only in -Fe, while in the presence of icronte supply slight decrese ws induced (-30%) (Tle 4). Western lot nlysis reveled n incresed level of the plsm memrne H + -ATPse protein in plnts grown under oth stress conditions. However, the increse ws more evident under Fe-free nutrient solution thn under icronte supply, confirming the results otined oth in vivo nd in vitro (see Fig. 4 & Tle 4). Polyclonl ntiodies rised ginst sorghum PEPC were lso used to ssess the presence of this enzyme in root solule extrcts nd to evlute the expression level under stress conditions. The Western lot results re shown in Fig. 5. In the control lne, the ntiody rected only ginst polypeptide with n pprent moleculr mss of 103 kd. However, in Fe-deficient condition, two polypeptides corresponding to 103 nd 108 kd, respectively, were detected, in greement with previous studies (De Nisi & Zocchi 2000). No pprecile nds were evident in the lne corresponding to root solule extrcts grown under icronte supply. These results re in good greement with the ctivities presented in Tle 4. Shikimte pthwy enzymes As shikimic cid is precursor in the iosynthesis of romtic compounds, nd consequently of phenolics, some enzymtic ctivities with key role in the shikimte H + -ATPse PEPC +Fe -Fe +FeBic Figure 5. Western lot nlysis of H + -ATPse nd phosphoenolpyruvte croxylse (PEPC) crried out on root plsm memrne preprtions nd root solule extrcts from plnts grown for 9 d in control conditions (+Fe), Fe deficiency (-Fe) nd in the presence of icronte (+FeBic), respectively. Plsm memrne or solule proteins (15 mg) were loded on discontinuous SDS-polycrylmide gel (8%). After SDS-PAGE, proteins were electrophoreticlly trnsferred to polyvinylidene difluoride (PVDF) memrne filters nd two different ntiser were used; one rised ginst the centrl domin of PM H + -ATPse of Aridopsis thlin (1:10 000) nd the second one rised ginst PEPC isoform of sorghum (1:1000). The experiment ws repeted three times with the sme results Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

9 Adptive strtegy to limited iron vilility Flvonoids Erythrose-4-Phosphte + PEP DAHPS (Δ 280 mg -1 prot) SK (nmol min -1 mg -1 prot) 3-Dehydroshikimte Shikimte PAL (nmol min -1 g -1 FW) Stilenes DAHP Shikimte-3-Phosphte Phenyllnine p-coumroyl CoA Phenylpropnoid esters SDH (nmol min -1 mg -1 prot) EPSPS (nmol Pi mg -1 prot) c Anthocynins Isoflvonoids Figure 6. Enzyme ctivities of shikimte pthwy in extrcts of roots from control (+Fe), Fe-deficient (-Fe) nd icronte (+FeBic) treted plnts. Dt re the mens SD (n = 9). In the cse of significnt differences (P < 0.05), vlues re mrked with different letters. DAHPS, 3-deoxy-D-rino-heptulosonte-7- phosphte synthse; EPSPS, 5-enol-pyruvyl-shikimte- 3-phosphte syntse; PAL, phenyllnine mmoni-lyse; SDH, shikimte dehydrogense; SK, shikimte kinse. Finlly, the PAL ctivity, the first enzyme in the phenylpropnoid iosynthetic pthwy, ws lso incresed significntly in oth stress conditions, ut more in +FeBic (+38 nd +92%, respectively). Oxygen consumption In order to otin n overview of mitochondril ctivity, the O 2 consumption rte of picl root segments ws determined. Figure 7 shows tht O 2 consumption rte decreses in roots of Fe-deficient (-40%) with respect to those from Fe-sufficient nd icronte-treted plnts. After KCN ddition, the totl O 2 consumption decresed y -17 nd -32% in control nd +FeBic roots, respectively, wheres in the sence of Fe the decrese ws lrger, -57%. The presence of SHAM, which inhiits the lterntive oxidse, cused greter decrese in the consumption of O 2 in control nd +FeBic conditions (-91 nd -94%, respectively), while in -Fe roots decrese ws c. 100% (Fig. 7). DISCUSSION A limited nutrient vilility generlly ffects plnt growth more thn photosynthesis, enling the lloction of photosynttes to the production of C-sed secondry metolites, such s phenolics (Lerdu & Coley, 2002). Accordingly, when root phenolics were quntified in Prietri plnts collected directly from the wlls (Tle 1), correltion etween their mount nd the ph of the sustrtes ws found. These dt might suggest tht plnts respond to the ph vrition through metolic rerrngement, modifying the lloction of cron skeletons etween primry nd secondry metolism. Interestingly, in the sme roots simultneous increse in the orgnic cid concentrtion occurs (Tle 1); these compounds re reported to e ccumulted nd exuded not only under Fe deficiency, ut lso under other nutrient deficiencies (Widodo et l. 2010; Lin et l. 2011). pthwy were ssyed (Fig. 6). In comprison with the control, Fe deficiency, especilly when induced y icronte, led to significnt increse in the ctivity of these enzymes. DAHPS, which is the key enzyme controlling the cron flow towrds phenolic metolism, incresed significntly when plnts were grown in the presence of icronte (+33% with respect to the control). The sme ws true for SDH ctivity (+41%) in greement with the fct tht plnts grown under icronte were chrcterized y higher phenolic contents. The SK ctivity, which converts the SDH s product in 3-phospho-shikimte, ws ffected in oth Fe-deficient conditions (Fig. 6). The ctivity of EPSP synthse in extrcts from -Fe roots did not significntly differ from tht found in the control (Fig. 6). On the contrry, in presence of icronte, the EPSP synthse ctivity incresed out 3-fold with respect to the +Fe tretment. nmol O 2 min -1 g -1 FW Fe IR KCN resistnt KCN+SHAM resistnt -Fe +FeBic +Fe c -Fe +FeBic +Fe -Fe +FeBic Figure 7. Oxygen consumption rte in excised picl root segments from plnts grown for 9 d in control condition (+Fe), Fe deficiency (-Fe) nd in the presence of icronte (+FeBic). Dt re the mens SD (n = 5). In the cse of significnt differences (P < 0.05), vlues re mrked with different letters. FW, fresh weight; IR, initil rte; SHAM, slicylhydroxmic cid Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

10 1180 S. Donnini et l. Susequently, to investigte growth nd physiologicl ehviour prticulrly concerning Fe uptke, rooted cuttings of Prietri were grown in hydroponic medium nd sumitted to direct (-Fe) or induced Fe deficiency in the presence of icronte (+FeBic). This lst condition would mimic the nturl environment where plnts grow showing stle phenotype. In fct, while the sence of Fe resulted in significnt decrese in the chlorophyll concentrtion cusing the clssicl symptoms of chlorosis, in the presence of icronte chlorophyll ws less ffected, showing wek or no symptoms of chlorosis (see Figs 1 & 3). However, under the ltter condition more significnt decrese in shoot growth ws oserved, proly s n dptive strtegy to mintin n dequte level of Fe (nd chlorophyll, s well) in the tissues (Fig. 2 & Tle 2), preserving the photosynthetic ctivity s it hs lredy een suggested for per plnts (Donnini et l. 2009). At root level, the -Fe condition induced the prolifertion of lterl roots (Moog et l. 1995; Dsgn et l. 2002; Jin et l. 2008), while the icronte supply induced shorter root system, with the ppernce of structures similr to proteoid roots (Purnell 1960), mintining the sme root mss s the control plnts. Proteoid roots, developing from secondry nd tertiry lterl roots, provide n enhnced surfce of contct etween plnt nd soil which is useful for the relese of nutrientsoluilizing compounds nd for the uptke of nutrients from the rhizosphere (Dell Orto et l. 2003; Lmers et l. 2003). The presence of such structures only in plnts grown with icronte suggests tht this should not e specific response to Fe deficiency ut to more generl condition of low nutrient vilility: indeed, P deficiency hs een found to e induced in clcreous soils s well (Guerinot & Yi 1994). In 9-dy-old Prietri, we found slight decrese in P concentrtion in oth Fe deficiency conditions (Tle 2). Preliminry findings otined y testing Prietri in the field prompted us to investigte oth the ccumultion nd exudtion of orgnic compounds. In prticulr, s suggested y Jin et l. (2007), phenolics could ply n importnt role in the moiliztion of Fe ccumulted in the highly negtively chrged cell wll, proly through chemicl cheltion nd reduction operted y phenolics themselves. We found n increse in the ccumulted nd exuded phenolics in oth conditions of Fe deficiency, which ws higher in the presence of icronte. However, the relese of phenolics into the exudtes in these conditions, which hs een ttriuted minly to proteoid roots tissues (Weisskopf et l. 2006), ppers to e scrce when compred with the mount ccumulted in the roots (Tle 1). Tle 3 shows the cpcity of these phenolics to reduce Fe(III)-EDTA in vitro, demonstrting tht they could effectively influence the reduction nd then the moility of Fe-cheltes oth in the poplst nd in the externl medium. The mjor Fe reduction efficiency detected under icronte supply might e scried to reltive undnce of specific compounds (results not shown), s the reducing cpcity hs een ttriuted to the presence of prticulr groups or doule onds in the moleculr structure (Bors et l. 1990). The nlysis of some key enzymes of the shikimte pthwy shows good correltion etween the concentrtion of phenolics nd n increse in the ctivities of severl enzymes in their iosynthetic pthwy. In fct, n enhnced mount of phenolic compounds, s well s increses in the ctivities of DAHP synthse, SDH, SK nd PAL, ws found in oth Fe-deficient conditions, nd the increse ws lrger under the icronte tretment with the exception of SK (see Tle 1 & Fig. 6). These results re lso in greement with dt y Ln et l. (2011) who find roust increse in the mount of enzymes linked to the phenylpropnoid pthwy induced y Fe deficiency. In the presence of icronte, we found lso n increse in the EPSP synthse, the sixth enzyme of the prechorismte pthwy, whose overexpression hs een ssocited to the tolernce to the hericide glyphoste, which locks the synthesis of romtic compounds (Amrhein et l. 1983; Comi, Sen & Stlker 1983; Reinothe, Nelles & Prthier 1991). Interestingly, resistnce to glyphoste hs een reported in Prietri (Protoppdkis 1985). When orgnic cids re mesured in oth roots nd exudtes, significnt increse ws detected in roots grown under oth stress conditions. Under Fe, ut lso P deficiency, orgnic cid excretion would increse plnt efficiency, through decrese in the ph of the medium nd consequently soluiliztion of Fe nd P. Our dt show tht in the presence of icronte, citric cid is the min exuded orgnic cid (Tle 1), s lredy reported in other species (Mssonneu et l. 2001; Jelli et l. 2010). On the other hnd, it hs een reported tht mlte is poor t moilizing micronutrients from the soil, while citrte is cple of moilizing significnt quntities (Jones & Drrh 1994). Furthermore, Lnglde et l. 2002) reported tht the rtio of mlte to citrte is higher only in the pex nd juvenile proteoid roots. P. diffus, Strtegy I plnt species (Dell Orto et l. 2003), responds to Fe deficiency y inducing the FC-R nd H + -ATPse ctivities (Fig. 4,e & Tle 4), the lst one ttriutle to n increse in the protein expression (Fig. 5). An increse in Strtegy I enzymtic ctivities ws lso oserved in +FeBic, lthough to minor extent, which could e ttriuted to the presence of Fe in the medium, which might in prt stisfy the plnt requirements. Interestingly, the results otined in vivo show tht when roots re grown with icronte, the reduction ctivity ppers widely distriuted long the roots (Fig. 4). We suggest tht for this tretment, the FC-R ctivity mesured in the plsmlemm frction nd known to e loclized minly t picl nd su-picl root zones (see -Fe tretment) could e only prt of the totl Fe reduction ctivity detected in vivo; the relese of reductnts (e.g. phenolics) could e responsile for the ctivity which ppers to e present long the whole root length. In the present work, n increse in the PEPC ctivity ws oserved only in -Fe; surprisingly, the icronte supply in the presence of Fe induces decrese in this ctivity nd the protein expression in root solule extrcts shown y Western lot nlysis confirms these results (Fig. 5) Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

11 Adptive strtegy to limited iron vilility 1181 Another intriguing result otined in Prietri is the different presence of polypeptides recting with the ntiody. In fct, only in the sence of Fe we found oth the 103 nd 108 KD s reported in the PEPC literture (see De Nisi nd Zocchi 2000 nd references therein). Phosphoenolpyruvte (PEP) is sustrte for the rections ctlyzed y oth PEPC nd DAHP synthse. We suggest tht in the presence of icronte, the PEP produced y glycolysis could e in prt chnnelled into the shikimte pthwy nd converted into phenolics, which re undnt in this condition. In murl hitt, the ph uffered to cuses precipittion of Fe long with other nutrients. In this condition, the Strtegy I ctivities might ecome indequte to ssure Fe supply if not y mens of n extreme effort y H + -ATPse ctivity to counterct the uffering effect of icronte. In this cse, the Fe pool stored in the root poplst could represent vlid extrcellulr reserve; the more plnt will e le to moilize such pools, the more it will e competitive in these growing conditions. We show tht P. diffus responds to the presence of icronte through n overproduction of phenolics nd orgnic cids. Furthermore, dt otined through n experimentl pproch in hydroponic culture suggest tht this ehviour could e relted to metolic shift involving prticulrly the PEP-consuming rections. In the sence of Fe, Prietri would need more energy for the ctivtion of the Strtegy I responses. However, in this condition, the primry source of energy (mithochondrion) is impired (Fig. 7; see lso Vigni et l. 2009). It hs een shown tht in Fe-deficient cucumer roots the rte of glycolysis nd the PEPC ctivity re incresed to produce the sustrtes useful to sustin FC-R nd H + -ATPse ctivities (De Nisi & Zocchi 2000; Espen et l. 2000; Zocchi 2006). The PEP produced could then e ddressed to the sustennce of iosynthesis of oth phenolics nd orgnic cids. In the presence of icronte, Fe is present in the externl nd poplstic solution even if in unville forms, nd the metolic choice of Prietri might e the ctivtion of the shikimte pthwy to produce phenolics, useful to Fe desorption from the cell wll. In the presence of Fe nd icronte, the mitochondril electron trnsport chin is unffected in comprison with the control (Fig. 7), remining the primry Figure 8. Schemtic representtion of metolic chnges occurring in roots of Prietri diffus growing in control conditions (+Fe), Fe deficiency (-Fe) nd in the presence of icronte (+FeBic). Dotted lines indicte the metolic pthwys nd Strtegy I ctivities in the control condition (+Fe). In -Fe nd +FeBic pnels, the rrow thickness would indicte the chnge in the metolic flux nd Strtegy I ctivities. FC-R, Fe 3+ -chelte reductse; IRT1, iron-regulted trnsporter 1; PEP, phosphoenolpyruvte; PEPC, phosphoenolpyruvte croxylse Blckwell Pulishing Ltd., Plnt, Cell nd Environment, 35,

12 1182 S. Donnini et l. mechnism to produce energy; furthermore, s the mitochondri re not significntly ffected, the TCA cycle could etter produce orgnic cids, explining the increse in these compounds seen in this condition. The diversion from primry metolism for prt of the PEP pool is in greement with the decrese in the PEPC ctivity oserved in the presence of Fe nd icronte. This enzyme is negtively regulted y L-mlte (Chollet, Vidl & O Lery 1996), whose concentrtion increses under this condition nd, s proposed y recent work (De Mrí et l. 2006), y shikimte, which hs een shown to exert puttive inhiitory effect on the PEPC, reinforcing the hypothesis. In conclusion, while Prietri grown in the sence of Fe shows metolic responses similr to other Strtegy I plnts growing in the sme condition, under +FeBic this species is le to diversify its response y incresing lterntive pthwys for Fe soluiliztion. In clcreous soils, where Fe is present ut scrcely solule nd more likely trpped in the cell wll, the ptitude of the secondry metolism to ssume relevnt role could e responsile for the Prietri s exceptionl efficiency. We summrize the results of this nd previous studies (Dell Orto et l. 2003) y proposing model for metolic rerrngement in Prietri (Fig. 8). ACKNOWLEDGMENTS We wish to thnk Dr M. Dell Orto, Dr G. Vigni nd Dr M. Whellens for continuous dvice nd for criticl reding of the mnuscript. We thnk Dr G. Vigni for the schemtic representtion of Fig. 8. We wish lso to thnk Dr R. Serrno (Universidd Politécnic, Vlenci, Spin) nd Dr J.Vidl (Université de Pris-Sud) for providing the ntiodies used in this work. REFERENCES Adí J., Vázquez S., Rellán-Álvrez R., El-Jendoui H., Adí A., Álvrez-Fernández A. & López-Millán A.F. (2011) Towrds knowledge-sed correction of iron chlorosis. Plnt Physiology nd Biochemistry 49, Amrhein N., Johrming D., Sch J. & Schulz A. 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