Gibberellins regulate iron deficiency-response by influencing iron transport and translocation in rice seedlings (Oryza sativa)

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1 nnls of otny 119: , 17 doi:1.19/o/mw5, ville online t Gierellins regulte iron defiieny-response y influening iron trnsport nd trnslotion in rie seedlings (Oryz stiv) oln Wng 1,, Hifng Wei 1,, Zhen Xue nd Wen-Ho Zhng 1,,, * 1 Stte Key Lortory of Vegettion nd Environmentl Chnge, Institute of otny, the Chinese demy of Sienes, eijing 19, PR Chin, Reserh Network of Glol Chnge iology, eijing Institutes of Life Siene, Chinese demy of Sienes, eijing 19, Chin, University of Chinese demy of Sienes, eijing 19, PR Chin nd Institute of otny, the Chinese demy of Sienes, eijing 19, PR Chin *For orrespondene. E-mil whzhng@is..n Reeived: 19 ugust 16 Returned for revision: 1 Otoer 16 Editoril deision: 1 Otoer 16 Pulished eletronilly: 8 Jnury 17 kground nd ims Gierellins (Gs) re lss of plnt hormones with diverse funtions. However, there hs een little informtion on the role of Gs in response to plnt nutrient defiieny. Methods To evlute the roles of Gs in regultion of Fe homeostsis, the effets of G on Fe umultion nd Fe trnslotion in rie seedlings were investigted using wild-type, rie mutnt (eui1) displying enhned endogenous G onentrtions due to defet in G detivtion, nd trnsgeni rie plnts overexpressing OsEUI. Key Results Exposure to Fe-defiient medium signifintly redued iomss of rie plnts. oth exogenous pplition of G nd n endogenous inrese of iotive G enhned Fe-defiieny response y exggerting folir hlorosis nd reduing growth. Iron defiieny signifintly suppressed prodution of G 1 nd G, the iologilly tive Gs in rie. Exogenous pplition of G signifintly deresed lef Fe onentrtion regrdless of Fe supply. Iron onentrtion in shoot of eui1 mutnts ws lower thn tht of WT plnts under oth Fe-suffiient nd Fe-defiient onditions. Ploutrzol, n inhiitor of G iosynthesis, llevited Fe-defiieny responses, nd overexpression of EUI signifintly inresed Fe onentrtion in shoots nd roots. Furthermore, oth exogenous pplition of G nd endogenous inrese in G resulting from EUI muttion inhiited Fe trnslotion within shoots y suppressing OsYSL expression, whih is involved in Fe trnsport nd trnslotion. Conlusions The novel findings provide ompelling evidene to support the involvement of G in medition of Fe homeostsis in strtegy II rie plnts y negtively regulting Fe trnsport nd trnslotion. Key words: Rie (Oryz stiv), Fe defiieny, strtegy II plnt, gierellins, Fe trnsport nd trnslotion. INTRODUCTION Iron (Fe) is n essentil nutrient required for plnt growth nd development. Iron vilility is often low in soils due to its low soluility, espeilly in lreous soils, leding to limited plnt growth (Romer nd lntr, ). Plnts hve evolved two ontrsting strtegies to ope with Fe defiieny in soils. Strtegy I ours in non-grmineous monoots nd diots, while strtegy II exists in grmineous monoots (Römheld nd Mrshner, 1986; Koyshi nd Nishizw, 1). Strtegy I plnts re distinguished y enhned ferri redutse tivity, idifition of the rhizosphere nd upregultion of Fe þ trnsporters upon exposure to Fe-defiient medium (Curie nd rit, ; Hell nd Stephn, ; Romer nd lntr, ). In ontrst, Strtegy II plnts often show enhned seretion of phytosiderophores (PSs), whih elong to the muginei id (M) fmily, to form Fe þ -M omplex in response to Fe defiieny (Koyshi nd Nishizw, 1). Ms re synthesized from three moleules of S-denosyl-methionine y three sequentil enzymes: niotinmine (N) synthse (NS), N minotrnsferse (NT) nd deoxymuginei id synthse (DMS). In rie, genes enoding these enzymes hve een identified (Inoue et l., ). The Fe þ -M is trnsported into root ells y memrne trnsporter of Yellow Stripe1 (YS1) in mize (von Wiren et l., 199; Curie et l., 1). For rie, the seretion of Ms from roots to the rhizosphere is medited y OsTOM1 (Nozoye et l., 11), nd the resulting Fe þ -M omplexes re tken up into root ells y OsYSL15, yellow-stripe like (YSL) trnsporter in the plsm memrne (Lee et l., 9). In ddition to the YSL system, rie plnts n lso quire Fe þ through IRT trnsporters, nd two genes enoding the Fe þ trnsporters, OsIRT1 nd OsIRT, hve een isolted (ughio et l., ; Ishimru et l., 6). OsYSL is rie metl-n trnsporter responsile for trnslotion of Fe nd Mn in shoots vi the phloem, nd it lso plys roles in the medition of trnsport of Fe from roots to shoots (Koike et l., ; Ishimru et l., 1). In ddition, severl trnsription ftors tht re involved in regultion of Fe homeostsis hve een isolted in rie. These inlude OsIRO, OsIRO, OsIDEF1, OsIDEF nd OsHLH1. For instne, OsIRO is synhronously expressed with genes involved in Fe homeostsis, nd responsile for rie growth nd yield in lreous soils (Ogo et l., 7). Despite identifition of numer of key genes involved in Fe homeostsis in rie, the overll signlling network ssoited with Fedefiieny responses remins lrgely to e disseted. Numerous studies hve shown tht plnt hormones ply importnt roles in responses to stresses ssoited with defiieny VC The uthor 17. 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 96 Wng et l. Gierellins re involved in Fe homeostsis in rie nd/or toxiity of minerl nutrients. For instne, uxin, ethylene, ytokinins nd nitri oxide (NO) hve een reported to e involved in the regultion of Fe defiieny-indued physiologil proesses in strtegy I plnts (Ivnov et l., 1). lthough there re reports inditing tht ethylene is not involved in the regultion of Fe-defiieny responses y strtegy II plnts (Romer et l., 1999; Romer nd lntr, ), Wu et l. (11) showed tht ethylene regultes the expression of genes ssoited with Fe quisition in rie plnts. s rie plnts hve oth strtegy I nd II systems for Fe quisition, it is likely tht, similr to strtegy I plnts, plnt hormones my lso ply regultory role in medition of Fe quisition y rie plnts. However, in ontrst to strtegy I plnts, there hs een limited informtion on the roles of phytohormones in response of strtegy II plnts to Fe defiieny. We reently demonstrted tht rssinosteroids re involved in Fe homeostsis in rie (Wng et l.,15). Gierellins (Gs) re one of the lssil plnt hormones with multiple funtions in the regultion of physiologil proesses ssoited with developmentl nd environmentl ues. For exmple, Jing et l. (7) reported tht G is involved in the regultion of response to phosphte defiieny in ridopsis. Mtsuok et l. (1) reported tht expression of mny genes ssoited with Fe-defiieny responses ws upregulted in G-defiient ridopsis mutnt, strtegy I plnt, y exogenous pplition of G. Very reently, Wild et l. (16) showed tht the sptil distriution of Gregulted DELL growth repressors in ridopsis roots ws dpted to the root system rhiteture nd the Fe-uptke mhinery to the plnt s Fe demnd. Given the differenes in Fe moiliztion nd trnsport etween strtegy I nd strtegy II plnts, whether nd how G plys role in Fe-defiieny responses in strtegy II plnts remin to e investigted. Studies on G funtion hve een gretly promoted y isoltion of numerous rie mutnts ssoited with G metolism nd signlling (Tong et l., 1). mong the mutnts, the reessive tll rie mutnt elongted uppermost internode (eui) exhiits rpid nd enhned elongtion of the internode nd inresed endogenous G onentrtions. The EUI gene enodes G-detivting enzyme, nd overexpression of EUI results in mrked inhiition of plnt growth (Luo et l., 6; Zhu et l., 6). In the present study, we used wild-type (WT), eui1 mutnt nd EUI overexpression line to evlute the role of G in the response of strtegy II rie plnts to Fe defiieny. We lso monitored the hnges in endogenous tive G levels in WT rie plnts in response to Fe defiieny. Our results show tht Fe defiieny signifintly inhiited G 1 nd G prodution in WT rie plnts, nd tht G negtively modultes Fe utiliztion in rie seedlings y reduing Fe trnsport nd trnslotion in shoots. MTERILS ND METHODS Plnt growth nd tretments Rie seeds of Zhonghu 1, ZS97, eui1 nd EUI-overexpression (EUI-OE) lines were surfe-sterilized y inution for min in 75 % ethnol, followed y 1 min in 1 % HgCl,nd then wshed thoroughly with sterile wter. Seeds of the eui1 mutnt nd EUI-OE were kindly provided y Professor Z. H. He t Shnghi Institutes for iologil Sienes, Chinese demy of Sienes. GC-MS nlysis showed tht eui1 plnts ontined muh higher level of G 1 nd G thn did wildtype plnts, nd the EUI-OE plnts hd n extremely dwrf phenotype (Zhu et l., 6). The sterilized seeds were soked in wter for h in the drk nd then trnsferred to nylon net floting on wter for 1 week. Then, the 7-d-old seedlings were trnsferred to nutrient solution ontining one-hlf-strength Kimur solution. The nutrient solution ontined the mronutrients (mm) (NH ) SO (18), MgSO 7H O(7), KNO (9), K SO (7), C(NO ) H O (18) nd KH PO (9), nd the mironutrients (mm) MnCl H O(9), H O (6), H MoO (6), ZnSO 7H O(77) nd CuSO 5H O (), supplied with 5 mm Fe (III)-EDT with ph of 55, nd the nutrient solution ph ws djusted dily nd ws renewed every d. The hydroponi experiments were rried out in growth room with 16-h light ( C)/8-h drk ( C) photoperiod, nd the reltive humidity ws ontrolled t pprox. 7 %. fter 1 week preulture, the rie plnts were exposed to Fe-suffiient [1 mm Fe (III)-EDT[ or Fe-defiient [ mm Fe (III)-EDT] full-strength Kimur solution with nd without 1 mm gierelli id (G )or1mm ploutrzol (PC) for 1week. Mesurement of hlorophyll onentrtion fter tretments with different levels of Fe nd G/PC supply for 7 d, the fully expnded youngest leves were used to determined totl hlorophyll onentrtion (SPD vlues) with portle SPD meter (SPD-5) (Zhejing Top Instrument Co., Ltd, Chin). Determintion of plnt growth nd nlysis of metl onentrtion fter tretments with different levels of Fe nd G/PC supply for 7 d, shoot length ws reorded y ruler, nd seedlings were hrvested nd seprted into leves, stems nd roots. Fresh iomss of different tissues ws mesured nd the tissues were then dried for d t 75 C for determintion of dry iomss. fter mesurements of iomss, lef, stem nd root smples were digested with onentrted nitri id nd hydrogen peroxide. The totl Fe onentrtions were determined y indutively oupled plsm emission spetrometry (ICP-OES). Determintion of endogenous iotive G onentrtions The endogenous onentrtions of iotive G 1 nd G in leves nd stems of WT rie plnts were determined y GC- MS following modifition of the protools desried y Wijynti et l. (1995). riefly, rie leves nd stems ( mg) were olleted, instntly frozen in liquid nitrogen nd ground with pestle. To etter preserve the Gs, the ground leves nd stems were dried under vuum (8 mr) t low temperture ( 5 C). The dried rie lef nd stem smples were weighed, reorded, extrted with MeOH/H O ¼ :1 ontining [ H ]G 1 nd [ H ]G s internl stndrds nd evported to dryness. The dried extrt ws re-dissolved in MeOH/

3 Wng et l. Gierellins re involved in Fe homeostsis in rie 97 H O ¼ :1 nd pssed through C18 Sep-pk olumn nd HPLC olumn of ZORX S-C18 devie (15 mm 6mm, 5lm). The frtions orresponding to G 1 nd G were olleted nd dried in entrifugl onentrtor. The G frtions were trimethylsilylted with N-methyl-N-trimethylsilylteifluoroetmide (MSTF) t 8 C for min. The smples were tken to dryness nd dissolved in hexne prior to injeting into fused sili glss pillry olumn (D-5, 5 mm m, 5 lm film thikness, J&W). Injetion nd interfe tempertures were 6 nd 8 C, respetively. The olumn temperture ws mintined t 8 Cformin,nd then inresed y 1 Cmin 1 to 18 C, followed y 6 C min 1 to 9 C. Eletron energy ws 7 ev. The identity of Gs ws verified y monitoring dignosti ions of oth endogenous nd deuterted Gs ording to the referene of Fernndez et l. (1997). The internl stndrds [ H] -G 1 (t. no. 91) nd [ H] -G (t. no. 51) were purhsed from OlChemIm Compny. Expression of genes enoding Fe uptke nd trnslotion Totl RN ws extrted from roots nd shoots of rie plnts sujeted to Fe or G tretments. Totl RN ws isolted using RNiso regent (Tkr) nd ws reverse-trnsried into first-strnd DN with PrimeSript RT regent Kit with gdn Erger (Tkr). Rel-time PCR ws performed in n optil 96-well plte with n pplied iosystems Stepone reltime PCR system. Eh retion ontined 75 ll of SYR Green Mster Mix regent, 5 ll of DN smples nd 6 ll of 1 mm gene-speifi primers in finl volume of 15 ll. The therml yle used ws s follows: 95 C for 1 min; yles of 95 C for s, 55 C for s nd 7 Cfor s. ll the primers used for quntittive RT-PCR re listed in Supplementry Dt Tle S1. The reltive quntifition method (DDCt) ws used to evlute quntittive vrition etween the replites exmined. The PCR signls were normlized to those of tin or rie polyuiquitin (RuQ1). Sttistil nlysis two-wy NOV ws onduted using the SS sttistil softwre. Signifint differenes etween tretments were evluted y lest signifint differene multiple rnge tests (P 5). RESULTS Effets of exogenous pplition of G nd G iosynthesis inhiitor on plnts To test whether G is involved in Fe-defiieny-indued hnges in physiologil proesses in rie plnts, the effets of exogenous pplition of G on growth nd hlorophyll (CHL) onentrtion of rie seedlings grown in Fe-suffiient nd Fedefiient medi were studied. n pprent hlorosis ws oserved in the newly formed leves of rie seedlings grown in Fe-defiient medium for 1 week (Fig. 1). ordingly, exposure of rie plnts to Fe defiieny led to signifint derese in CHL onentrtion (Fig. 1). Furthermore, the hlorosis eme more evident y pplition of G to Fe-defiient rie seedlings (Fig. 1). In ontrst, pplition of G hd no pprent effet on lef hlorosis in rie seedlings grown in Fe-suffiient medium (Fig. 1). In ddition to exogenous G pplition, we further evluted the effet of PC, n inhiitor of G iosynthesis, on plnt growth nd CHL onentrtion. Iron-defiieny-indued hlorosis eme less visile when PC ws dded to Fe-defiient medium (Fig. 1). ordingly, we found tht tretment with PC led to signifint inrese in CHL onentrtion of Fe-defiient seedlings, nd tht the sme PC tretment lso enhned CHL onentrtion in Fe-suffiient rie seedlings (Fig. 1). There ws signifint intertion etween Fe tretments nd G-relted tretments in CHL onentrtion (P ¼ 11). In ddition to CHL onentrtion, Fe defiieny lso inhiited shoot elongtion nd plnt growth (Fig. ). pplition of G signifintly inresed shoot length under oth Fesuffiient nd Fe-defiient onditions (Fig. ). Sine G redued CHL onentrtion y out % (Fig. 1), ut inresed shoot length (Fig. C), we further ssessed the effets of G on lef, stem nd root growth under Fe-suffiient nd Fe-defiient onditions. Similr to CHL onentrtion, exposure to Fedefiient medium led to signifint redution in lef, stem nd root iomss. In ontrst, G pplition hd no effet on iomss of lef, stem nd root of Fe-suffiient rie seedlings (Fig. D). In ontrst to lef iomss, iomss of stem nd root ws not ffeted y G under Fe-defiient onditions (Fig. C, D). pplition of PC signifintly suppressed shoot length nd redued plnt iomss, irrespetive of Fe supply (Fig. ). For exmple, iomss of lef nd stem ws signifintly redued y PC tretment under oth Fe-suffiient nd Fe-defiient onditions (Fig., C). However, the mgnitude of the inhiition in Fe-defiient seedlings ws less thn tht in Fe-suffiient seedlings. Endogenous iotive G onentrtions in lef nd stem of WT plnts were redued y Fe defiieny The oservtions tht exogenous pplition of G enhned Fe-defiieny-indued lef hlorosis of rie plnts imply tht G levels my ply n importnt role in Fe sttus in plnt. To test if Fe sttus lso ffets iotive G prodution, we monitored the effet of Fe defiieny on onentrtions of the two iotive Gs (G 1 nd G ) in leves nd stems of WT plnts. s shown in Fig., exposureofwtrieplntstofedefiient medium led to signifint redutions in G 1 nd G onentrtions in leves nd stems (Fig. ). This result onfirms tht Fe sttus is involved in G iosynthesis in rie plnts. The eui1 mutnt nd EUI-OE line differed in their responses to Fe defiieny EUI1 enodes ytohrome P5 monooxygense tht epoxidizes G in novel detivtion retion in rie, nd muttion of EUI onfers higher tive G onentrtion in plnt in the eui1 mutnt (Luo et l., 6; Zhu et l., 6). To further investigte the intertion mong G nd Fe, rie mutnt eui1 nd trnsgeni rie line EUI-OE were used. No signifint differenes in CHL onentrtion mong the mutnt, EUI-OE line

4 98 Wng et l. Gierellins re involved in Fe homeostsis in rie /G /PC /G /PC 5 Control +G +PC SPD vlue 1 Tretments FIG. 1. Effet of iron, G nd PC tretments on lef hlorophyll onentrtions. One-week-old rie plnts (Zhonghu 1) pre-ultured with Fe (III)-EDT were exposed to Fe-suffiient [1 mm Fe (III)-EDT] or Fe-defiient [ mm Fe (III)-EDT] onditions with or without 1 mm G or 1 mm PC for 1 week. Chlorophyll onentrtions of the fully expnded youngest lef were mesured using SPD hlorophyll meter. Dt re mens6s.d. (n ¼ 1). Different pitl letters indite signifint differene (P 5) etween þfe nd tretment for the tree regimes (ontrol, þ G nd þpc), nd different lowerse letters denote signifint differene (P 5) mong the three tretments under the sme Fe onditions. nd WT plnts were deteted when grown in Fe-suffiient medium (Fig. ). Exposure of these plnts to Fe-defiient medium led to signifint redution in folir CHL in WT, eui1 nd EUI-OE plnts, with the mgnitude of redution following the order eui1 > WT > EUI-OE (Fig. ), inditing tht rie plnts with higher endogenous G onentrtions re more sensitive to Fe defiieny. These results re lso in greement with those of exogenous pplition of G nd PC. EUI muttion signifintly inresed shoot length under oth Fe-suffiient nd Fe-defiient onditions, nd overexpression of

5 Wng et l. Gierellins re involved in Fe homeostsis in rie 99 Shoot length (m per plnt ) C Stem iomss (mg per plnt ) Control +G +PC Lef iomss (mg per plnt ) D Root iomss (mg per plnt ) Control +G +PC Tretments Tretments FIG.. Effet of Fe, G nd PC tretments on plnt growth. One-week-old rie plnts (Zhonghu 1) pre-ultured with Fe (III)-EDT were exposed to Fe-suffiient [1 mm Fe (III)-EDT] or Fe-defiient [ mm Fe (III)-EDT] onditions with or without 1 mm G or 1 lm PC for 1 week. Shoot () length nd iomss of different orgns (, C nd D) were mesured. Dt re mens6s.d (n ¼ 9 for shoot length, nd n ¼ 5 for iomss). Tretments nd sttistil nlysis re s detiled in Fig. 1. this gene inhiited shoot growth (Fig. ). Compred to the WT plnt, muttion nd overexpression of this gene signifintly inresed nd deresed respetively the iomss of lef nd stem regrdless of Fe supply (Tle 1). Muttion of eui1 hd no impt on root iomss, while EUI-OE redued root iomss y 5 % (Tle 1). iomss of lef nd stem in WT, eui1 nd EUI-OE plnts ws signifintly redued y exposure to Fe-defiient medium, while root iomss of WT, eui1 mutnt nd EUI-OE plnts ws similr (Tle 1). Fe quisition ws redued y oth exogenous G pplition nd muttion of the EUI gene In ddition to CHL nd plnt growth, we evluted the effets of G on Fe onentrtions in leves, stems nd roots. Exposure of rie seedlings to Fe-defiient medium led to mrked redution of Fe onentrtions in leves, stems nd roots of WT, eui1 nd EUI-OE plnts (Fig. 5). Exogenous pplition of G redued Fe onentrtion in lef, while it hd no effet on stem Fe onentrtion (Fig. 5). y ontrst, exogenous pplition of G mrkedly inresed Fe onentrtion in roots under oth Fe-suffiient nd Fe-defiient onditions (Fig. 5). These results my imply tht G is involved in regultion of Fe trnslotion from roots to leves. Compred to WT plnts, muttion of EUI led to signifint redution in Fe onentrtions in leves nd stems, while Fe onentrtion in roots ws not ffeted y the muttion, irrespetive of Fe supply (Fig. 5). Overexpression of EUI in rie plnts signifintly inresed Fe onentrtions in leves, stems nd roots under oth Fe-suffiient nd Fe-defiient onditions (Fig. 5). There ws signifint intertion etween Fe tretments nd Grelted tretments in root Fe onentrtion (P ¼ 7). Exogenous G pplition redued folir CHL onentrtions in eui1 mutnt nd EUI-OE plnts Exogenous pplition of G enhned Fe-defiienyindued folir hlorosis in WT plnts. We further exmined the responses of the eui1 mutnt nd EUI-OE plnts to exogenous G pplition under oth Fe-suffiient nd Fe-defiient onditions. Similr to WT plnts, G pplition hd no impts on folir CHL onentrtions in the eui1 mutnt nd EUI- OE plnts under Fe-suffiient onditions (Fig. 6, ), while exogenous pplition of G signifintly redued folir CHL onentrtions in the eui1 nd EUI-OE plnts under Fe-defiient onditions (Fig. 6C, D). Furthermore, the mgnitude of redution in folir CHL onentrtion ws higher in the eui1 mutnt thn in WT plnts. These results suggest tht folir CHL onentrtion my e negtively orrelted with endogenous G onentrtions in leves under Fe-defiient onditions. Expression ptterns of Fe defiieny-responsive genes were regulted y G Given the importnt role of trnsription ftor OsIRO in Fe homeostsis in rie (Ogo et l., 7), we ompred the

6 95 Wng et l. Gierellins re involved in Fe homeostsis in rie Endogenous tive G level (ng g 1 f. wt) Lef SPD vlue 1 WT eui1 EUI-OE Endogenous tive G level (ng g 1 f. wt) Stem FIG.. Effet of Fe defiieny on endogenous iotive G prodution in lef () nd stem (). One-week-old rie plnts of WT (ZS 97) pre-ultured with Fe (III)-EDT were exposed to Fe-suffiient [1 mm Fe (III)-EDT] or Fe-defiient [ mm Fe (III)-EDT] onditions for 1 week. iotive G in lef nd stem ws mesured. Dt re mens6s.d (n ¼ or 5). Mens with different letters denote signifint differene etween þfe nd tretments (P 5). responses of this gene in roots of WT, eui1 mutnt nd EUI-OE plnts to exogenous pplition of G under Fe-suffiient nd Fe-defiient onditions t the trnsriptionl level. Neither exogenous pplition of G nor muttion of the EUI gene hd ny impt on the expression level of OsIRO under Fesuffiient onditions, ut overexpression of EUI signifintly depressed the expression of OsIRO under Fe-suffiient onditions (Fig. 7). Iron defiieny up-regulted the expression of OsIRO in WT, eui1 mutnt nd EUI-OE plnts (Fig. 7). Moreover, exogenous pplition of G nd Fe defiieny hd n dditive effet on the expression of OsIRO (Fig. 7). Compred to WT, the EUI muttion inhiited the expression of OsIRO under Fe-defiient onditions, nd EUI-OE did not ffet the expression of OsIRO under Fe-defiient onditions (Fig. 7). In ddition to OsIRO, we lso evluted the effet of G on the expression of genes relted to M iosynthesis nd Fe þ -M uptke in roots of WT, eui1 mutnt nd EUI-OE plnts, inluding OsNS1, OsNS, OsNT1, OsDMS1 nd OsYSL15. SimilrtoOsIRO, exposure to Fe-defiient medium led to up-regultion of these genes (Fig. 7 F). The expression levels of OsNS1, OsNS nd OsDMS1 in WT plnts were not ltered y ddition of G to the Fe-suffiient nd Fedefiient medi, nd only OsNT1 ws up-regulted y G under Fe-defiient onditions (Fig. 7 E). In ontrst, the expression of OsYSL15 in WT plnts ws enhned y G Shoot length (m per plnt) Fe Tretments pplition regrdless of Fe supply (Fig. 7F). Muttion of EUI enhned expression of OsNS1, OsNS nd OsNT1, while the trnsript levels of OsDMS1 nd OsYSL15 remined unhnged y the muttion under Fe-suffiient onditions (Fig. 7 F). The expression of OsNS1 ws suppressed y the EUI gene muttion under Fe-defiient onditions. Overexpression of EUI led to depressed expression of ll these genes under Fe-suffiient onditions (Fig. 7 F). Tretment of EUI-OE plnts with Fe defiieny up-regulted expression of OsNS1 nd OsNS, while it suppressed expression of OsYSL15 with the expression of remining genes unhnged (Fig. 7 F). There ws n extremely signifint intertion etween Fe tretments nd G-relted tretments in the expression of OsIRO, OsNS1, OSNS nd OsYSL15 of root (P < 1). OsYSL enodes n Fe-N trnsporter nd plys n importnt role in the trnslotion of Fe in shoots (Ishimru et l., 1). We monitored the expression ptterns of OsYSL in WT, eui1 mutnt nd EUI-OE plnts under oth Fe-suffiient nd Fe-defiient onditions. Iron defiieny up-regulted the expression of OsYSL in shoots of WT, eui1 nd EUI-OE plnts (Fig. 8). The expression of OsYSL in shoots ws signifintly Fe FIG.. Lef hlorophyll nd shoot length of WT (ZS 97), eui1 nd EUI-OE rie plnts grown in Fe-suffiient nd Fe-defiient onditions. One-week-old rie plnts of WT, eui1 nd EUI-OE pre-ultured with Fe (III)-EDT were exposed to Fe-suffiient [1 mm Fe (III)-EDT] or Fe-defiient ( mm Fe (III)-EDT] onditions for 1 week. Lef hlorophyll () nd shoot length () were mesured. Dt re mens6s.d (n ¼ 1). Different pitl letters denote signifint differene (P 5) for the three plnt mterils (WT, eui1 nd EUI-OE) etween þfe nd tretments. Different lowerse letters denote signifint differene (P 5) for the three plnt mterils within the sme Fe tretments.

7 Wng et l. Gierellins re involved in Fe homeostsis in rie 951 TLE 1. iomss of different plnt orgns of WT (ZS 97), eui1 nd EUI-OE rie plnts grown in Fe-suffiient nd Fe-defiient onditions Plnt orgn Tretment Plnt orgns f. wt iomss (mg per plnt) WT eui EUI-OE Lef þfe * 969 * Stem þfe 87766* 18876* Root þfe Dt re mens6s.e. *Signifint differene etween þfe nd tretment within the sme ultivr t the level of P 5. Mens with different letters re signifintly different (P 5) within three ultivrs t the sme Fe tretments. depressed y G pplition nd EUI muttion under oth Fesuffiient nd Fe-defiient onditions (Fig. 8). Overexpression of EUI signifintly up-regulted the expression of OsYSL in shoots under Fe-suffiient onditions, ut down-regulted its expression under Fe-defiient onditions (Fig. 8). There ws n extremely signifint intertion etween Fe tretments nd G-relted tretments in the expression of OsYSL of shoot (P < 1). DISCUSSION There is inresing evidene highlighting the involvement of G in the medition of ioti stress responses (Ueguhi- Tnk et l., 7; Ymguhi, 8). However, few studies hve speifilly investigted the role of G in responses of plnts to defiieny in minerl nutrients. There re limited reports demonstrting the possile involvement of G in the ontrol of Fe homeostsis in plnts. Mtsuok et l. (1) reported tht exogenous G ltered expression of Fe uptkerelted genes in the gox1/gx doule mutnt, Gdefiient mutnt of ridopsis under Fe-suffiient onditions. However, the uthors did not evlute the effets of G on physiologil proesses ssoited with Fe quisition nd utiliztion in WT plnts. In reent study, Wild et l. (16) provide experimentl evidene showing rosstlk etween G signlling nd Fe quisition in ridopsis, highlighting the ritil roles of G in sensing nd dpting to Fe defiieny in plnts (von Wiren nd ennett, 16). Sine rie nd ridopsis differ sustntilly in their responses to Fe defiieny, whether nd how G regultes Fe quisition in rie plnts remin to e evluted. In the present study, we explored the roles of G in the medition of Fe quisition under oth Fe-suffiient nd Fe-defiient onditions y investigting the effets of exogenous G on growth, Fe umultion, CHL onentrtion nd genes involved in Fe homeostsis of WT rie seedlings. In ontrst to ridopsis, our results reveled tht n exogenous supply of G enhned folir hlorosis of Fe-defiient rie seedlings, nd tht G redued CHL onentrtion regrdless of Fe supply levels. Furthermore, we demonstrted tht exogenous pplition of G inhiited Fe umultion in lef, ut not in stem nd root, under oth Fesuffiient nd Fe-defiient onditions. Similr to exogenous G pplition, the eui1 mutnt with higher endogenous iotive G onentrtion ws more sensitive to Fe defiieny thn WT plnts, while trnsgeni EUI-OE rie plnts were less responsive to Fe defiieny thn WT plnts. nother importnt finding in the present study is tht the onentrtions of iologilly tive G 1 nd G in leves nd stems of WT rie plnts were signifintly redued upon exposure to Fe-defiient medium (f. Fig. ). Finlly, we demonstrted tht oth exogenous pplition of G nd inreses in endogenous G onentrtion due to disruption of G metolism inhiited the expression of OsYSL in shoots (Fig. 8), whih in turn my suppress the unloding of Fe from the phloem to mesophyll ells, thus resulting in enhned folir hlorosis. These results lerly indite tht G plys importnt roles in Fe trnsport nd trnslotion in strtegy II rie plnts. G is well-known phytohormone tht promotes plnt growth (Ymguhi, 8). For instne, G tretment n signifintly inrese shoot length of rie (Luo et l., 6; Li et l., 11). In the present study, we showed tht oth exogenous nd endogenous G enhnement promoted shoot length under oth Fe-suffiient nd Fe-defiient onditions, nd tht the G synthesis inhiitor PC nd overexpression of EUI inhiited shoot length nd plnt iomss (Figs 1 nd ). These oservtions re in greement with those reported in the literture. G inresed shoot length, ut oth exogenous G pplition to WT nd n endogenous inrese of G in the eui1 mutnt enhned Fe-defiieny-indued lef hlorosis. Growth stimultion n inrese the Fe demnd of plnts. In this study, exogenous pplition of G enhned shoot length under oth Fesuffiient nd Fe-defiient onditions. However, G pplition hd no impt on shoot iomss, while lef iomss ws even redued y G under Fe-defiient onditions (Fig. ). These results suggest tht G-indued lef hlorosis my not result from high Fe demnd y plnts due to G-dependent growth stimultion. Compred to WT, n endogenous inrese of tive G in the eui1 mutnt stimulted shoot length nd iomss. Lef Fe onentrtion of eui1 ws signifintly redued under oth Fe-suffiient nd Fe-defiient onditions. Under Fe-suffiient onditions, suffiient mount of Fe ws present in the growth medium, nd the redution in folir Fe onentrtion is unlikely to result from inreses in Fe demnd y plnts. In ddition to Fe, we lso mesured onentrtions of other mironutrient nd mronutrient metl ions in leves of WT nd eui1 under oth Fe-suffiient nd Fe-defiient onditions. Our results showed tht neither exogenous nor endogenous inreses in G ltered folir Mn, Zn, Mg nd K

8 95 Wng et l. Gierellins re involved in Fe homeostsis in rie Lef Fe (µg g 1 lef f. wt) Stem Fe (µg g 1 stem f. wt) C Root Fe (µg g 1 root f. wt) WT onentrtions under oth Fe-suffiient nd Fe-defiient onditions (Supplementry Dt Fig. S1). There ws n inrese in folir Mn nd Zn onentrtions upon exposure to Fe-defiient medium. These results re in line with those reported in the literture (Morrissey nd Guerinot, 9). However, G did not hve n impt on folir Mn nd Zn onentrtions regrdless of Fe levels in the growth medium. These results highlight tht the effet of G is speifi to Fe. CHL is mjor omponent of hloroplsts nd hs een used s n inditor of Fe sttus in plnts (Grzino et l., ). + Fe eui1 Tretments WT/G Fe EUI-OE FIG. 5. Iron onentrtions of different plnt orgns of WT (ZS 97), eui1 nd EUI-OE rie plnts grown in Fe-suffiient nd Fe-defiient onditions. Oneweek-old rie plnts of WT, eui1 nd EUI-OE pre-ultured with Fe (III)-EDT were exposed to Fe-suffiient [1 mm Fe (III)-EDT] or Fe-defiient [ mm Fe (III)-EDT] for one week. Iron onentrtions in different orgns were mesured. Dt re mens6s.d (n ¼ ). Different pitl letters denote signifint differene (P 5) for the three plnt mterils etween þfe nd tretment. Different lowerse letters denote signifint differene (P 5) for the three plnt mterils under the sme levels of Fe supply. There re reports showing tht G plys role in CHL iosynthesis in ridopsis (Jing et l., 1). However, unlike Fe, the effet of G on CHL iosynthesis my e indiret (Jing et l., 1). Moreover, very high onentrtion of exogenous G (1 mm) ws used in the study of Jing et l. (1), while muh lower exogenous G onentrtion (1 mm) ws pplied in the present study. lthough muh higher endogenous tive G level ws deteted in the eui1 mutnt, its CHL onentrtion ws not ffeted y exogenous pplition of G under Fe-suffiient onditions (Fig. 6). These results indite tht G-enhned lef hlorosis my not result from its effet on CHL metolism. In the present study, in ontrst to WT, folir Fe onentrtions of WT in the presene of exogenous G nd of the eui1 mutnt were redued y % under Fe-defiient onditions (Fig. 5), whih my ount for the ggrvted folir hlorosis nd redution in lef iomss under Fe-defiient onditions. Correspondingly, folir CHL nd Fe onentrtions were signifintly inresed in the dwrfed EUI-OE plnts. The effet of G on Fe onentrtions of WT ridopsis ws not determined, nd shoot Fe onentrtion in G-defiient doule mutnt (gox1/gox) ws inresed y 17-fold while exogenous pplition of G redued Fe onentrtion in shoot of the mutnt to level omprle to tht in the WT (Mtsuok et l., 1). However, Wild et l. (16) reported tht Fe onentrtion in the G-defiient g1- mutnt ws lower thn tht in WT ridopsis plnts. The differenes in Fe onentrtion etween the G-defiient doule mutnt gox1/ gox nd G-defiient g1- mutnt my result from the smpling protools used in the two studies. For exmple, in the study using the gox1/gox doule mutnt, shoot nd root re seprted, while in g1- plnts, the whole plnt ws used. In the present study, we found tht exposure of WT plnts to Fe-defiient medium led to signifint redution in onentrtions of G 1 nd G, the iologilly tive Gs in WT plnts (f. Fig. ). In ridopsis, Fe-depleted plnts exhiited lower levels of iotive G nd its preursors thn Fesuffiient plnts (Wild et l., 16). In mize, endogenous G levels in shoots were redued y Fe defiieny, ut tretment with G iosynthesis inhiitors hd no impt on shoot Fe onentrtions (Sekimoto et l., 1). Different tretment times, plnt speies nd tissues used to mesure Fe onentrtions in these studies my ount for the different results. For exmple, neither Fe defiieny nor G iosynthesis inhiitors were found to ffet plnt height nd fresh weight, nd shoot Fe onentrtion ws determined y mixing leves nd stems (Sekimoto et l., 1). In the present study, tretments with Fe defiieny nd G iosynthesis inhiitor ltered plnt growth, nd exogenous pplition of G only redued lef Fe onentrtion, ut it hd no effet on stem Fe onentrtion (f. Fig. 5), suggesting tht exogenous G possily plys importnt roles in lef Fe homeostsis. In ontrst to exogenous G pplition to WT plnts, Fe onentrtion in stems ws lso redued nd enhned y muttion nd overexpression of EUI, respetively. This is possily due to differene in distriution of exogenously pplied G nd iosyntheti G of plnts within leves nd stems. For exogenous pplition, G ws dded in the hydroponi solution, nd G ws tken up y roots nd trnsported to shoots y trnspirtion. Sine trnspirtion rte in lef is higher thn in stem, greter mount of G is expeted to umulte in lef, leding to more umultion

9 Wng et l. Gierellins re involved in Fe homeostsis in rie 95 C SPD vlue 1 WT WT/G eui1 eui1/g EUI-OE EUI-OE/G WT eui1/g WT/G EUI-OE WT WT/G eui1 eui1/g EUI-OE EUI-OE/G eui1 D WT WT/G EUI-OE/G eui1/g EUI-OE SPD vlue 1 e eui1 EUI-OE/G tretments tretments FIG. 6. Effet of G pplition on lef hlorophyll of WT (ZS 97), eui1 nd EUI-OE plnts grown under Fe-suffiient nd Fe-defiient onditions. One-week-old rie plnts of WT, eui1 nd EUI-OE pre-ultured with Fe (III)-EDT were exposed to Fe-suffiient [1 mm Fe (III)-EDT] or Fe-defiient [ mm Fe (III)-EDT] with or without 1 mm G for 1 week. Lef hlorophyll ws mesured. Dt re mens6s.d (n ¼ 1). Tretments nd sttistil nlysis re s detiled in Fig. 5. of G in lef thn in stem, nd stronger responses of lef to exogenous G pplition thn stem in WT plnts. For eui1 nd EUI-OE plnts, euse EUI ws minly expressed in rpidly elongting nd/or dividing tissues, inluding the shoot pil meristems, the divisionl (interlry meristem) nd elongting zones of internodes, nodes of n elongting stem nd pnile (Zhu et l.,6), G levels in lef nd stem of the eui1 mutnt nd EUI-OE plnts my e omprle, resulting in lower nd higher Fe onentrtions of lef nd stem in eui1 mutnt nd EUI-OE plnts thn WT plnts, respetively. Tken together, these results indite tht G plys importnt nd omplited roles in Fe trnslotion nd distriution, depending on plnt speies, G level, Fe sttus, growth stge nd different orgns. In rie seedlings, lef Fe sttus is likely to e negtively orrelted with endogenous G levels in plnts. Supplementtion of G further ggrvted lef hlorosis in the eui1 mutnt, nd redued CHL onentrtion of EUI-OE plnts under Fe-defiient onditions, whih orroortes this onlusion (Fig. 6). In this study, we found tht Fe onentrtion in root ws signifintly inresed y suppression of endogenous G regrdless of Fe supply, inditing tht G is possily involved in the regultion of Fe uptke y roots. The oservtions tht the eui1 mutnt nd WT plnts exhiited omprle Fe onentrtions in their roots under Fe-suffiient nd Fe-defiient onditions my suggest tht n endogenous inrese in G level hs no effet on Fe onentrtions in rie roots, ut exogenous pplition of G signifintly inresed Fe onentrtion in roots of WT plnts under Fe-suffiient onditions. In ridopsis, root Fe onentrtion in the doule mutnt gox1/gox ws lower thn in WT plnt root, ut Fe onentrtion in the doule mutnt ws inresed out % in root nd redued y out % in shoot in response to exogenous G pplition (Mtsuok et l., 1). These results suggest tht the effet of G on Fe onentrtion in roots my e more omplited thn in shoots. oth exogenous G pplition nd endogenous inhiition of G prodution y overexpression of EUI enhned Fe onentrtion in rie roots, ut the underlying mehnisms my e different. For EUI-OE plnts, higher root Fe onentrtion ws orrelted with higher lef nd stem Fe onentrtion. However, for exogenous G pplition, higher root Fe onentrtion ws possily indued y suppressing Fe trnsport from roots to shoots. Iron defiieny enhnes Fe uptke nd trnslotion from roots to shoots (Vert et l., ). Our results imply tht G is likely to e involved in the regultion of Fe uptke, trnsport nd trnslotion within rie plnts. The trnsription ftor OsIRO is essentil in modulting expression of genes ssoited with Fe homeostsis in rie plnts (Koyshi nd Nishizw, 1). Iron defiieny indued up-regultion of OsIRO expression, thus tivting the expression of genes responsile for Fe uptke nd trnslotion in roots. Our results re in line with those reported in the literture. In the present study, we found tht G hd no speifi effets on the expression of OsIRO under Fe-suffiient nd Fe-defiient onditions, nd tht the expression of genes relted to Fe uptke ws not in greement with the expression pttern of OsIRO (Fig. 7). In ridopsis, exogenous pplition of G to the G-defiient ridopsis mutnt gox1/gox up-regulted expression of Fe uptke-relted genes nd inresed root Fe onentrtion, while shoot Fe onentrtion in the mutnt ws signifintly redued y G (Mtsuok et l., 1). These results suggest tht G possily prtiiptes in regulting the expression of

10 95 Wng et l. Gierellins re involved in Fe homeostsis in rie Root OsIRO D 1 Root OsNS1 Reltive expression 1 WT WT/G eui1 EUI-OE Reltive expression 8 6 WT WT/G eui1 EUI-OE 5 Root OsNS E 5 Root OsINT1 Reltive expression 1 Reltive expression 1 C 5 Root OsDMS1 F 5 Root OsYSL15 Reltive expression 1 + Fe Tretments Fe Reltive expression 1 Tretments FIG. 7. Quntittive RT-PCR nlysis of Fe uptke-relted genes in roots. Dt re mens6s.e. of three iologil replites. Different pitl letters denote signifint differene (P 5) for WT, WT/G, eui1 nd EUI-OE etween þfe nd tretments. Different lowerse letters denote signifint differene (P 5) for WT, WT/G, eui1 nd EUI-OE under the sme Fe tretments. Fe-uptke-relted genes t trnsriptionl or posttrnsriptionl levels, nd tht G negtively regultes Fe trnsport from roots to shoots. Very reently, Wild et l. (16) demonstrted tht G signlling hs tissue-speifi dul funtion in the dpttion to Fe-defiient environment in ridopsis. When Fe vilility is redued, DELLs re umulted in the root meristem, therey restrining root growth nd progressively exluded from epiderml ells in the root growth differentition zone (Wild et l., 16). The exlusion of DELL from the site of Fe quisition relieves FIT from DELL-dependent inhiition, thus enhning Fe uptke (Wild et l., 16). In our study, whole roots were used to detet gene expression, mking it impossile to monitor tissue-speifi expression of genes. Wild et l. (16) further demonstrted tht the rie DELL, SLR1, n intert with IRO, suggesting tht similr G-signlling mehnism my exist in strtegy I ridopsis nd strtegy II rie plnts in response to Fe defiieny. There hve een numerous reports showing differenes etween ridopsis nd rie in Fe quisition nd G signlling. For instne, five DELL proteins hve een identified in ridopsis (Dviere nd hrd, 1), while only one DELL protein, SLR1, ws identified in rie (Iked et l., 1). Furthermore, in ridopsis, onstitutive FIT overexpression does not indue downstrem genes, nd FIT forms

11 Wng et l. Gierellins re involved in Fe homeostsis in rie 955 Reltive expression WT WT/G Shoot Os YSL eui1 EUI-OE Tretments FIG. 8. Quntittive RT-PCR nlysis of OsYSL in shoots. Dt re mens6s.e. of three iologil replites Tretments nd sttistil nlysis re s detiled in Fig. 7. heterodimers with HLH8 nd/or HLH9 under Fe-defiient onditions, nd the omplexes n tivte the trnsription of FRO nd IRT1 in root epiderml ells (Yun et l., 8). In rie, no similr interting prtner hs een identified, nd IRO n diretly regulte expression of vrious genes relted to Fe utiliztion nd genes involved in the methionine yle in strtegy II rie plnts (Koyshi nd Nishizw, 1). Therefore, it is oneivle tht G nd its signlling my hve different modes to regulte Fe quisition in rie nd ridopsis. Further studies iming to eluidte the moleulr mehnisms y whih G modultes uptke, trnsport nd trnslotion of Fe in rie plnts re wrrnted. OsYSL hs proved to e involved in the phloem trnsport of Fe in shoots (Koike et l., ). Our results showed tht oth exogenous nd endogenous inreses in G levels inhiited the expression of OsYSL in shoots (Fig. 8). The down-regultion of OsYSL my suppress the unloding of Fe from the phloem to mesophyll ells, nd redue internl Fe vilility in shoots, thus resulting in enhned lef hlorosis. However, the inhiition y G is round 15-fold under Fe-defiient onditions, nd EUI-OE even depressed its expression. Sine trnslotion of Fe within shoots remins lrgely to e determined (Koyshi nd Nishizw, 1), the depression of OsYSL my prtly explin the phenomenon, nd other mehnisms need to e explored in future studies. CONCLUSIONS We provide experimentl evidene in support of involvement of G in medition of responses of strtegy II rie plnts to Fe defiieny. More speifilly, we demonstrte tht G is involved in the modultion of Fe homeostsis in rie plnts y negtively regulting Fe trnsport from roots to shoots, nd trnslotion within shoots. Further studies iming to eluidte the moleulr mehnisms responsile for the effet of uptke, trnsport nd trnslotion of Fe in rie plnts y G, nd the ssoited G-signlling trnsdution pthwys, re wrrnted. SUPPLEMENTRY DT Supplementry dt re ville online t nls.org nd onsist of the following. Tle S1: the primers used for quntittive RT-PCR. Figure S1: effet of exogenous nd endogenous G inreses on Mn, Zn, Mg nd K onentrtions of lef under Fe-suffiient nd Fe-defiient onditions. CKNOWLEDGEMENTS This work ws supported y the Ntionl Siene Foundtion of Chin (1677 nd 11159) nd Stte Key Lortory of Vegettion nd Environmentl Chnge. We thnk Professor Z. He for the gift of eui1 mutnt nd EUI-OE plnts. LITERTURE CITED ughio N, Ymguhi H, Nishizw NK, Nknishi H, Mori S.. Cloning n iron-regulted metl trnsporter from rie. Journl of Experimentl otny 5: Curie C, rit JF.. Iron trnsport nd signling in plnts. nnul Review of Plnt iology 5: Curie C, Pnviene Z, Loulergue C, Dellport SL, rit JF, Wlker EL. 1. 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Rie plnts tke up iron s n Fe þ -phytosiderophore nd s Fe þ. The Plnt Journl 5: 5 6. Ishimru Y, Msud H, shir K, et l. 1. Rie metl-niotinmine trnsporter, OsYSL, is required for the long-distne trnsport of iron nd mngnese. The Plnt Journl 6: Ivnov R, rumrov T, uer P. 1. Fitting into the hrsh relity: regultion of iron-defiieny responses in diotyledonous plnts. Moleulr Plnt 5: 7. Jing CF, Go XH, Lio LL, Hrerd NP, Fu XD. 7. Phosphte strvtion root rhiteture nd nthoynin umultion responses re modulted y the Gierellin responses re modulted y the gierellin-dell signling pthwy in ridopsis. Plnt Physiology 15: Jing XS, Li HY, Wng T, et l. 1.Gierellin indiretly promotes hloroplst iogenesis s mens to mintin the hloroplst popultion of expnded ells. The Plnt Journl 7: Koyshi T, Nishizw NK. 1. Iron uptke, trnslotion, nd regultion in higher plnts. nnul Review of Plnt iology 6: Koike S, Inoue H, Mizuno D, et l.. OsYSL is rie metlniotinmine trnsporter tht is regulted y iron nd expressed in the phloem. 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