Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat

Biochem. J. (2002) 367, 491–497 (P in ed in G ea B i ain) 491 Cloning o hio edoxin h educ ase and cha ac e iza ion o he hio edoxin educ ase– hio edoxin hsys em om whea An onio J. SERRATO, Juan M. PE ; REZ-RUIZ and F ancisco J. CEJUDO1 Ins i u o de Bioquı!mica Vege al y Fo osı!n esis, Cen o de In es igaciones Cien ı! icas Isla de la Ca uja, A da Ame ! ico Vespucio s/n, 41092 Se illa, Spain Thio edoxins ha e ubiqui ous p o eins educed by NADPH– hio edoxin educ ase (NTR). They a e able o educe disulphides in a ge p o eins. In monoco s, hio edoxins haccumula e a high le el in seeds and show a p edominan localiza ion in he nucleus o seed cells. These esul s sugges ha he NTR– hio edoxin hsys em p obably plays an impo an ole in seed physiology. To da e, he s udy o his sys em in monoco s is limi ed by he lack o in o ma ion abou NTR. In he p esen s udy, we desc ibe he cloning o a ull-leng h cDNA encoding NTR om whea (T i icum aes ium). The polypep ide deduced om his cDNA shows close simila i y o NTRs om A abidopsis, con ains FAD- and NADPH-binding domains and a disulphide p obably in e ac ing wi h he disulphide a he ac i e si e o hio edoxin h. Whea NTR was exp essed in Esche ichia coli as a His- agged p o ein. The abso p ion spec um o he pu i ied ecombinan p o ein is ypical o la oenzymes. Fu he mo e, i showed NADPH-dependen hio edoxin h e- INTRODUCTION Thio edoxins a e ubiqui ous p o eins wi h molecula mass o 12–13 kDa, which a e ound in bac e ia, unicellula euka yo es, and plan and animal cells. These p o eins ha e a conse ed ac i e si e, WCGPC, con aining a edox disulphide b idge [1]. Thio edoxins a e able o educe speci ic disulphide b idges in a ge p o eins, hence egula ing he ac i i y o hese p o eins. Th ee hio edoxin ypes ha e been desc ibed in plan s [2]. Two o hem, hio edoxins mand , encoded by nuclea genes, a e loca ed in he chlo oplas and a e educed by e edoxin hio- edoxin educ ase using pho osyn he ic educing powe . These hio edoxins educe speci ic disulphide b idges and, in his way, egula e he ac i i y o enzymes o he Cal in cycle. The e o e hio edoxins play an essen ial ole in he con ol o pho osyn he ic ca bon me abolism [3]. The hi d ype, hio edoxin h, is ound in he cell cy oplasm o mos plan o gans. Reduc ion o hio edoxin hdepends on NADPH and is ca alysed by NADPH– hio e- doxin educ ase (NTR) [2,4]. In he las ew yea s, hio edoxin h sys ems speci ic o mi ochond ia ha e been iden i ied in yeas [5], mammal cells [6,7] and A abidopsis haliana [8]. NTR is a la oenzyme ha ca alyses he ans e o elec ons om NADPH, ia FAD and a edox-ac i e disulphide, o he disulphide b idge o hio edoxins h. Like hio edoxins h, NTR is widely dis ibu ed in p oka yo ic and euka yo ic cells; howe e , wo g oups o NTR ha e e ol ed wi h di e en molecula and kine ic cha ac e is ics [4]. P oka yo es ha e a lowe -molecula - Abb e ia ions used: DTNB, 5,5h-di hiobis-(2-ni obenzoic acid); IPTG, isop opyl β-D- hiogalac oside; NTR, NADPH– hio edoxin educ ase; TRXh, hio edoxin h. 1To whom co espondence should be add essed (e-mail jcejudo!us.es). duc ion ac i i y, hus con i ming ha he cDNA clone epo ed in he p esen s udy encodes whea NTR. Using he His- agged NTR and TRXhA (whea hio edoxin h), we success ully econ- s i u ed he whea NTR– hio edoxin hsys em in i o, as shown by he insulin educ ion assay. A polyclonal an ibody was aised agains whea NTR a e immuniza ion o abbi s wi h he pu i ied His- agged p o ein. This an ibody e icien ly de ec ed a single polypep ide o he co esponding molecula mass in seed ex ac s and i allowed he analysis o he pa e n o accumula ion o NTR in di e en whea o gans and de elopmen al s ages. NTR shows a wide dis ibu ion in whea , bu , su p isingly, i s accumula ion in seeds is low, in con as wi h he le el o hio edoxins h. Key wo ds: Esche ichia coli, ge mina ion, hio edoxin mu an , T i icum. mass NTR, which consis s o wo iden ical subuni s (app ox. 35 kDa) o ming a homodime in i s na i e o m. The c ys al s uc u e o NTR om Esche ichia coli con ains h ee delin- ea ed domains in each o he subuni s, he FAD, NADPH and cen al domain [9]. In he con e sion in o he educed o m, he NADPH domain o a es wi h espec o he FAD domain [10]. In con as , NTR om mammal cells consis s o a homodime o subuni s wi h a la ge molecula mass (app ox. 55 kDa). The human enzyme con ains he unusual selenocys eine as he penul ima e esidue a he C- e minus [11,12], which is ac i e in he ca alysis [13]. A peculia ea u e o he hio edoxin hsys em in plan s is he la ge numbe o genes encoding o hio edoxin h, a leas i e in A abidopsis [14], which a e in ol ed in mul iple unc ions, as shown by unc ional complemen a ion o yeas mu an s [15]. In ce eals, hio edoxin his abundan in seeds. Since hio edoxin h inac i a es inhibi o s o hyd oly ic enzymes [16] and ac i a es se ine p o eases a ea ly s ages o ge mina ion [17], i has been p oposed ha hey play an impo an ole as a signal o ce eal seed ge mina ion. Fu he analysis o hio edoxins hin ge mina ing and de eloping whea seeds e ealed hei accumu- la ion in he nuclei o aleu one and scu ellum cells [18], sugges ing he possibili y ha hese p o eins in e ac wi h nuclea ac o s, as is he case in mammal and yeas cells [19,20]. The cu en e idence poin s o he hypo hesis ha he hio edoxin hsys em is an impo an componen o monoco seed biology. Howe e , he unde s anding o his sys em in seeds is limi ed, because no #2002 Biochemical Socie y 492 A. J. Se a o, J. M. Pe ! ez-Ruiz and F. J. Cejudo desc ip ion o ce eal NTR has been epo ed so a . To ou knowledge, he only NTR cloned om plan s is om A. haliana [21]. The c ys al s uc u e o his enzyme was es ablished, so ha he e ia y s uc u e and kine ic pa ame e s a e known [22]. Howe e , no hing is known abou he pa e n o exp ession o NTR in A abidopsis. We we e in e es ed in de e mining he unc ion o he NTR– hio edoxin hsys em in ce eal seeds. Fo ha pu pose, we ha e isola ed a ull-leng h cDNA clone om whea encoding a deduced polypep ide wi h close simila i y o NTR. The p o ein, exp essed in E.coli, is a la oenzyme show- ing NTR ac i i y. In addi ion, we ha e aised a polyclonal an i- body agains he whea enzyme, which was used o analyse he pa e n o exp ession o NTR in whea plan s and seeds. MATERIALS AND METHODS Plan ma e ial Whea (T i icum aes ium c . Chinese Sp ing) plan s we e g own in a g eenhouse unde con olled condi ions on a mix u e o pea e miculi e (3:1). De eloping seeds we e ha es ed a 18 days pos an hesis (18 d.p.a.), ozen in liquid ni ogen and s o ed a k80 mC un il used. Ma u e seeds we e allowed o ge mina e a oom empe a u e on il e pape soaked wi h wa e unde s e ile condi ions o 4 days, and he aleu one laye , scu ellum and s a chy endospe m we e ca e ully dissec ed. Shoo s and oo s o Wes e n-blo analysis we e dissec ed om 2–4-day- old seedlings, and ma u e lea es om 7-week-old plan s. All eagen s we e o analy ical g ade and we e pu chased om Sigma. Whea NTR cDNA cloning A cDNA lib a y (app ox. 300000 plaque- o ming uni s) con- s uc ed in λg 11 wi h polyadenyla ed [poly(A)+] RNA om whea aleu one cells was sc eened wi h an i-NTR polyclonal an ibodies aised agains he spinach enzyme (kindly p o ided by D F. J. Flo encio) as desc ibed in [23]. The ou posi i e clones ob ained we e pu i ied and he sizes o hei cDNA inse s we e analysed. A e subcloning in pGEM ec o s (P omega), inse s we e sequenced in bo h s ands. To ob ain he ull-leng h sequence, he oligonucleo ide (5h-GACCGCGACTGCCACCG- 3h) was designed o pe o m apid ampli ica ion o cDNA ends using he Ma a hon cDNA syn hesis ki (ClonTech) and o al RNA om di e en whea issues. A ull-leng h cDNA was ob ained, cloned in pGEM ec o and sequenced in bo h s ands using he T7 Sequenase 2.0 DNA sequencing ki (USB, Cle eland, OH, U.S.A.). Exp ession o NTR in E. coli and p oduc ion o polyclonal an ibodies Whea NTR was exp essed in E.coli as a His- agged polypep ide. To ha end, he coding sequence was ampli ied om he cDNA using he oligonucleo ide 5h-GAGAGGATCCATGGAGGA- GGCGGCCG-3h, which added a BamHI si e (unde lined) a he pu a i e ini ial Me esidue, and he oligonucleo ide 5h-GAGAAAGCTTTAGTCAATCAGTCTTCCCTTCC-3h, which added a HindIII si e (unde lined) a he 3h-end. The PCR agmen was hen diges ed wi h BamHI and HindIII and subcloned in pQE-30 exp ession ec o (QIAexp ess exp ession sys em; Qiagen). The esul ing plasmid, e med pQE-TaN , was in oduced in E.coli XL1-Blue, which was ou inely g own in he p esence o 2%(w ) glucose o a oid exp ession o he whea p o ein be o e induc ion. Exp ession o he ecombinan p o ein was induced by incuba ion o ans o med E.coli XL1-Blue cells o 3 h a 30 mC in glucose- ee Lu ia–Be ani medium supple- men ed wi h 0.1 mM isop opyl β-- hiogalac oside (IPTG). The o e exp essed p o ein was pu i ied by Cu#+a ini y ch oma o- g aphy in p e-packed Hi-T ap a ini y columns (Pha macia, Uppsala, Sweden) acco ding o he manu ac u e ’s ins uc ions. Aliquo s o he pu i ied p o ein we e used o immunize abbi s a he Se ice o Animal P oduc ion (Uni e si y o Se ille, Spain). The immune se um was used o pu i y an i-NTR by P o ein A ch oma og aphy be o e use. No signal was de ec ed when Wes e n blo s we e p obed wi h he p e-immune se um. Cons uc ion o hio edoxin hC53S (Cys53 Se ) mu an and i s exp ession in E. coli The Cys&$ esidue o hio edoxin hac i e si e was mu a ed o se ine. Si e-di ec ed mu agenesis was pe o med by PCR using he His- agged TRXhA cDNA as empla e [18]. Two oligo- nucleo ides we e designed complemen a y o he sequence o be mu a ed, namely C53S-A (5h-CATGGTGTGGACCATCCCG- CATCATGGCTC-3h) and C53S-B (5h-GAGCCATGATGCG- GGATGGTCCACACCATG-3h), including a single change (unde lined), which esul ed in he eplacemen o he Cys&$ esidue by Se . The PCR (1 cycle a 94 mC o 1 min; 16 cycles a 94 mC o 30 s, a 55 mC o 1 min and a 68 mC o 14 min) was pe o med wi h P u polyme ase (S a agene). The PCR p oduc was hen diges ed wi h DpnI o 1 h a 37 mC o elimina e he me hyla ed empla e DNA. This DNA was used o ans o m E. coli XL1-Blue. The esul ing mu an cDNA was sequenced in bo h s ands o check o he co ec in oduc ion o he mu a ion and ha no addi ional mu a ions we e gene a ed du ing he p ocess. Wes e n-blo analysis Plan issues we e ozen in liquid ni ogen and g ound wi h a mo a and pes le. C ude ex ac s we e p epa ed om he g ound issue wi h ex ac ion bu e [50 mM T is HCl (pH 7.9) 0.2 mM EDTA 0.5 mM PMSF]. The homogenized mix u e was cen i- uged o 20 min a 15000 g. The esul ing supe na an con- s i u ed he cell- ee ex ac and was s o ed a k20 mC. P o ein samples we e subjec ed o SDS PAGE (10%gel) as epo ed in [24] and elec o ans e ed o a ni ocellulose shee . The shee was hen soaked in 15 mM T is HCl (pH 7.4) con- aining 0.2 M NaCl (bu e A) and 5%(w ) d y powde ed milk a oom empe a u e o a leas 1 h. A e o e nigh incuba- ion a 4 mC wi h P o ein A-pu i ied an i-NTR (dilu ed 1:1000 in bu e A), shee s we e washed ou imes o 15 min each wi h bu e A con aining 0.1%( ) Tween 20. Shee s we e hen incuba ed o 1 h in he same bu e con aining a ini y-pu i ied goa an i-( abbi IgG) alkaline phospha ase conjuga e dilu ed o 1:30000. Memb anes we e subsequen ly washed in bu e A. The eac ion was isualized by imme sion in a de eloping solu ion [20 ml o 100 mM T is HCl (pH 9.5), 5 mM MgCl#, 100 mM NaCl, 3 mg o 5-b omo-4-chlo o-3-indolyl phospha e and 6 mg o Ni o Blue Te azolium]. NTR and hio edoxin ac i i y assays NTR ac i i y was de e mined by he educ ion o 5,5h-di hiobis- (2-ni obenzoic acid) (DTNB) by he me hod o Holmg en and Bjo $ ns ed [25]. NTR ac i i y was also assayed by i s abili y o educe hio edoxin h, which educes insulin disulphides using he u bidime ic assay me hod as desc ibed p e iously [18]. E.coli hio edoxin was pu chased om Sigma. #2002 Biochemical Socie y 493Thio edoxin educ ase om monoco s RESULTS AND DISCUSSION Isola ion o a cDNA clone encoding NTR om whea A whea cDNA lib a y cons uc ed wi h poly(A)+RNA isola ed om aleu one cells o 1-day-imbibed seeds was sc eened wi h polyclonal an ibodies aised agains NTR om spinach lea es. Fou posi i e clones we e ob ained and pu i ied. All o hem con ained sho inse s, app ox. 300 bp, showing a high deg ee o simila i y o he C- e minus o NTR om A. haliana [21]. To ob ain he ull-leng h cDNA, an oligonucleo ide was designed o pe o m apid ampli ica ion o cDNA ends using RNA isola ed om di e en whea seedling issues as empla e. A 1045 bp cDNA clone was ob ained om oo RNA (EMBL Nucleo ide Sequence Da abase accession no. AJ421947). This cDNA con- ained a single open eading ame encoding a deduced poly- pep ide o 331 esidues, wi h an expec ed molecula mass o 34.903 kDa and pI 5.79. The sequence o he deduced polypep ide and he alignmen wi h o he NTR sequences is shown in Figu e 1. The whea enzyme shows he highes le el o simila i y o NTRs om A abidopsis. I con ains a pu a i e FAD-binding domain o med by wo mo i s (ma ked wi h * in Figu e 1): he mo i GxGxxA ( esidues 17–22, whea numbe ing), a he N- e minal egion o he p o ein, and he mo i TxxxxVFAAGD ( esidues 283–293) a he C- e minus. These wo mo i s o ming he FAD-binding domain a e conse ed in NTR om plan s and unicellula euka yo es. Howe e , he mo i GxGxxA is eplaced by GxGxxG in he E.coli enzyme (Figu e 1). An NADPH-binding domain (GxGxxA, esidues 164–169) was also ound in he whea NTR (indica ed by a owheads in Figu e 1), which is conse ed in all he NTRs compa ed he e. Finally, he ac i e si e o he whea NTR (ma ked wi h a box in Figu e 1) con ains a disulphide b idge ( esidues 145 and 148, whea numbe ing) po en ially in e ac ing wi h he hio edoxin hac i e si e WCGPC [18]. The NTR ac i e si e p esen s a pe ec ma ch when compa ed wi h he mo e closely ela ed NTRs, bu he whea enzyme shows a peculia i y, he conse a i e change o Th ( esidue 143), which is eplaced by he Se esidue in NTR om he o he sou ces (Figu e 1). In he A abidopsis enzyme, his Se esidue and Asp"$* (A abidopsis numbe ing) bind ia a wa e molecule o N-5 o he isoalloxazine ing [22]. I is no expec ed ha he Th esidue a ha posi ion in he whea enzyme will ha e any signi ican e ec on NTR ac i i y. In summa y, he p o ein deduced om he cDNA desc ibed in his epo con ains he cha ac e is ics o bac e ial and plan NTRs. A phylogene ic ee was cons uc ed compa ing NTR sequences om di e en sou ces (Figu e 2). The whea enzyme appea s g ouped wi h wo NTRs om A abidopsis ( e med A. haliana A and B espec i ely) in a b anch including NTRs om unicellula euka yo es. The hi d pu a i e NTR iden i ied in he A abidopsis genome ( e med A. haliana C), al hough g ouped wi h hese NTRs, shows a weake ela ionship. The e o e he phylogene ic analysis e eals a close ela ionship o dico and monoco NTRs and a weak ela ionship wi h mammal NTRs. O he wo g oups o NTR ha ha e e ol ed [4], i is clea ha he plan and lowe euka yo es belong o he bac e ial- ype g oup. Exp ession o whea NTR in E. coli and cha ac e iza ion o he ecombinan p o ein To ob ain de ini i e e idence ha he whea cDNA epo ed he e encodes o an enzyme wi h NTR ac i i y, he coding sequence was subcloned in pQE-30 exp ession ec o , gene a ing he plasmid pQE-TaN , so ha whea NTR was p oduced as a N- e minus His- agged polypep ide. This app oach was chosen o acili a e he pu i ica ion o he ecombinan whea NTR, a oiding possible con amina ion wi h he E.coli enzyme, which shows simila ea u es and migh be co-pu i ied wi h he whea enzyme. E.coli XL1-Blue was ans o med wi h plasmid pQE- TaN and wi h pQE-30 ec o as con ol. SDS PAGE analysis o soluble p o eins ex ac ed om E.coli (pQE-TaN ) ha had been induced wi h IPTG shows he o e exp ession o a poly- pep ide wi h he expec ed molecula mass o 36.4 kDa, including he His ag (Figu e 3A, lane 2), which was no p esen in E.coli cells ans o med wi h he exp ession ec o wi hou inse (Figu e 3A, lane 1). Abou hal o he exp essed p o ein was ound in he pelle ( esul s no shown), indica ing ha pa o his p o ein is p obably o ming inclusion bodies. Since he soluble supe na an con ained a signi ican amoun o he whea enzyme, we used his soluble ex ac as he s a ing ma e ial o pu i y he His- agged NTR by Cu#+a ini y ch oma og aphy. This p o- cedu e yielded a highly pu i ied enzyme, based on SDS PAGE analysis (Figu e 3A, lane 3). In spi e o losing pa o he enzyme in inclusion bodies, he o e all yield o his p ocess was 1.6 mg l o cul u e. To es whe he he whea NTR is an FAD enzyme, as sugges ed by he sequence compa ison, we pe o med an abso p ion spec um o he pu i ied p o ein (Figu e 3B). The spec um is ypical o la op o eins showing cha ac e is ic ab- so p ion maxima a 270, 378 and 454 nm, clea ly showing ha he exp essed p o ein is a la oenzyme. To con i m ha his la oenzyme co esponded o whea NTR, we es ed i s abili y o educe whea hio edoxin hini o. Fo ha pu pose, we used he u bidime ic insulin p ecipi a ion assay [25]. Whea hio edoxin h(pu i ied His- agged TRXhA [18]) was able o educe insulin, p o ided ha NTR and NADPH we e p esen in he eac ion mix u e (Figu e 4). The eloci y o he eac ion, as shown by he inc ease in A'&!, was dependen on he concen a ion o NTR a sa u a ing concen a ions o TRXhA and NADPH and did no occu in he absence o ei he NTR o NADPH. The e o e, hese esul s con i m ha he cloned cDNA encodes o whea NTR, a la oenzyme, which e icien ly educes whea hio edoxin husing NADPH as elec on dono . Fu he mo e, hese esul s show ha he whea NTR– hio edoxin hsys em is e icien ly econs i u ed in i o using pu i ied His- agged componen s. Fu he e idence ha he whea NTR– hio edoxin his unc ional in i o was ob ained by he cons uc ion o he hio edoxin hmu an C53S, in which he Cys esidue (Cys&$) o he whea TRXhA ac i e si e was mu a ed o he Se esidue. This mu a ion comple ely abolished he unc ionali y o he sys em, as obse ed when he wild- ype hio edoxin hwas eplaced by C53S mu an polypep ide in he insulin educ ion assay (Figu e 5). Kine ic pa ame e s o whea NTR To de e mine he Km o NADPH and NADH, we used he DTNB educ ion assay [25]. Al hough whea NTR was able o use NADH, he Kmo he enzyme o NADPH is h ee o de s o magni ude lowe (Table 1). Since NTR showed a simila Vmax wi h bo h NADPH and NADH (Table 1), we conclude ha , as expec ed, he in ioelec on dono o whea NTR is mos p obably NADPH. The u bidime ic insulin p ecipi a ion assay e ealed ha he Km alue o whea NTR o whea hio edoxin hwas 7.6 µM (Table 1), which is highe han 1 µM p e iously epo ed o he A abidopsis enzyme [21]. Sequence simila i y and he in i o cha ac e iza ion o he NTR– hio edoxin hsys em allow us o p opose a eac ion mechanism simila o hose p e iously de- sc ibed o E.coli and A abidopsis sys ems [22]. Acco ding o his model, i should be expec ed ha whea NTR is able o in e ac #2002 Biochemical Socie y 494 A. J. Se a o, J. M. Pe ! ez-Ruiz and F. J. Cejudo Figu e 1 Mul iple alignmen o hio edoxin educ ase om di e en sou ces The p o ein sequence deduced om he whea cDNA (Ta; accession no. AJ421947) was aligned wi h closely simila NTR sequences sea ched in EMBL and SWISSPROT da abases using he p og am BioEdi sequence alignmen edi o [30]. The accession numbe s a e: A. haliana, A NTRA (AAB86519), A NTRB (CAB54874), A NTRC (AAB84351); Schizosaccha omyces pombe, Schp (CAA17692); Saccha omyces ce e isiae, Sce 1 (P29509), Sce 2 (AAA64747.1); Neu ospo a c assa, Neuc (P51978); mi ochond ial NTR om S. ce e isiae, mi Sce (P38816); Pencillium ch ysogenum, Pench (P43496) and E. coli (P09625). The FAD-binding domain is indica ed by * and he NADPH-binding domain by >, and he Cys esidues a he ac i e si e ma ked wi h a box a e in bold ace. #2002 Biochemical Socie y 495Thio edoxin educ ase om monoco s Figu e 2 The phylogene ic ee o NTRs om di e en sou ces The phylogene ic ee was cons uc ed using he p og am CLUSTALW (www.ebi.ac.uk/clus alw/), wi h NTR sequences sea ched in EMBL and SWISSPROT da abases. The accession numbe s a e: A. haliana (A, AAB86519; B, CAB54874; C, AAB84351); S. pombe (CAA17692); S. ce e isiae (1, P29509; 2, AAA64747.1; mi ochond ial, P38816); N. c assa (P51978); P. ch ysogenum (P43496); Zymomonas mobilis (AF124757); Haemophilus in luenzae (P43788); Ye sinia pes is (NPI404967); E. coli (P09625); Neisse ia meningi idis (NPI284253); Bacillus sub ilis (NPI391359); The mo oga ma i ima (NPI228678); S ep ococcus mu ans (JC2311); The moplasma olcanium (NPI110989); Plasmodium alcipa um (CAA60574); Mus musculus (AAH13688); Homo sapiens (1, XPI049211; 2, AAH07489; 3, S66677; 4, NPI006431). and educe E.coli hio edoxin. The esul s p esen ed in Table 1 show ha his is he case; howe e , whea NTR shows a poo eac i i y wi h he bac e ial enzyme, as e ealed by he highe Km (36 µM) and lowe Vmax. The esul s epo ed he e o he whea NTR a e in ag eemen wi h p e iously epo ed da a o he A abidopsis enzyme [21], con i ming ha , in spi e o he s uc u al ela ionship be ween plan and E.coli NTRs, he plan enzyme shows a poo eac i i y wi h bac e ial hio edoxin. These esul s u he suppo he close ela ionship o A abidopsis and whea NTR. Exp ession pa e n o NTR in whea To analyse he pa e n o accumula ion o NTR in di e en whea issues and de elopmen al s ages, an an ibody was aised agains whea NTR. This an ibody e icien ly de ec ed amoun s as low as 2 ng o he pu i ied His- agged enzyme in Wes e n-blo analysis (Figu e 6A). In p o ein ex ac s om ma u e whea seeds, he an ibody de ec ed a single polypep ide wi h he expec ed molecula mass o 35 kDa (Figu e 6A). I should be no ed ha he sligh ly di e en elec opho e ic mobili y is due o he His ag o he ecombinan enzyme. Acco ding o he in ensi y o he band de ec ed in he Wes e n-blo analysis, we could es ima e a con en o app ox. 1–2 ng µg o p o ein ex ac s om ma u e whea seeds. The Wes e n-blo analysis o he same amoun o p o ein ex ac s om di e en issues shows a high accumula ion o Figu e 3 Pu i ica ion and abso p ion spec um o whea His- agged NTR p oduced in E. coli (A) Analysis o an SDS/PAGE gel s ained wi h Coomassie B illian Blue R-250 ha was loaded wi h he supe na an o ex ac s (20 µg o p o ein) om pQE-30 wi hou inse (lane 1), o pQE- TaN (lane 2) a e IPTG induc ion. Lane 3, pu i ied His- agged TaNTR (1 µg o p o ein) a e Cu2+a ini y ch oma og aphy. (B) Abso p ion spec um o he pu i ied His- agged TaNTR. The spec um was eco ded using a 5 µM NTR solu ion in 100 mM K-phospha e bu e (pH 7.0) a oom empe a u e. Figu e 4 Insulin educ ion assay o he whea NTR– hio edoxin hsys em The incuba ion mix u e con ained he indica ed concen a ion o NTR, 3 µM His- agged TRXhA, 0.5 mg/ml insulin, 150 mM NADPH, 2 mM EDTA and 100 mM po assium phospha e bu e (pH 7.0). The basal line was also ob ained when NADPH was omi ed om he eac ion mix u e. #2002 Biochemical Socie y 496 A. J. Se a o, J. M. Pe ! ez-Ruiz and F. J. Cejudo Figu e 5 E ec o eplacemen o he wild- ype hio edoxin hby he C53S mu an on he ac i i y o he NTR– hio edoxin hsys em Assays we e pe o med in 100 mM po assium phospha e bu e (pH 7.0) con aining 50 nM NTR, 150 mM NADPH, 0.5 mg/ml insulin, 2 mM EDTA and 3 µM o ei he wild- ype o mu an C53S hio edoxin h, as indica ed. Table 1 Kine ic pa ame e s o whea NTR Whea hio edoxin hco esponds o His- agged TRXhA and was pu i ied as desc ibed in [18]. E. coli hio edoxin was pu chased om Sigma. The Kmand Vmax alues o hio edoxin we e de e mined using he insulin educ ion assay. The Kmand Vmax alues o NADPH and NADH we e de e mined using he DTNB assay. Subs a e Km Vmax (µmol :min−1:mg o p o ein−1) NADPH 0.4 µM 1.43 NADH 0.6 mM 1.38 Thio edoxin h7.6 µM 1.43 Thio edoxin (E. coli )36µM 0.42 NTR in ege a i e issues, in shoo s and oo s om 2–4-day-old seedlings, and in lea es om ma u e plan s (Figu e 6B). The con en o NTR was lowe in seeds, ei he in ma u e seeds (MS in Figu e 6B) o in de eloping seeds ha es ed a 18 d.p.a. (DS in Figu e 6B). An ea lie epo has desc ibed a ela i ely in a iable con en o NTR in he endospe m o whea g ains a e ge mina ion [26]. We ha e analysed he con en o NTR in he di e en issues o ge mina ing seeds; o ha pu pose, he aleu one laye , scu ellum and s a chy endospe m om seeds up o 4 days o imbibi ion we e ca e ully dissec ed. Figu e 6(C) shows a highe con en o NTR in he scu ellum, which sligh ly inc eased du ing he ime a e imbibi ion. The aleu one cells con ained a lowe con en o NTR, which simila ly inc eased a e ge mina ion (Figu e 6C). Su p isingly, he an ibody de ec ed wo bands in p o ein ex ac s om he s a chy endo- spe m. The band co esponding o he 35 kDa subuni showed a sligh dec ease, p obably due o he p o eoly ic ac i i y in he s a chy endospe m du ing he ime a e imbibi ion [27]. I is no clea whe he he uppe band de ec ed by hese an ibodies is ac ually NTR. This polypep ide shows a clea inc ease in s a chy endospe m ex ac s om seeds a e 2 days o imbibi ion, which hen p og essi ely disappea ed (Figu e 6C). Since his poly- pep ide was no de ec ed in ex ac s om aleu one o scu ellum cells, i is unlikely ha i is syn hesized and sec e ed om hese issues. I is equally unlikely ha i is syn hesized in he s a chy endospe m, which is a dead issue in seeds a e ge mina ion [28]. Figu e 6 Wes e n-blo analysis o NTR accumula ion in whea (A) P o ein ex ac s (15 µg) om ma u e whea seeds and inc easing amoun s (2, 10 and 50 ng) o he pu i ied His- agged TaNTR. (B) An equal amoun o ex ac s (15 µg o p o ein) om de eloping seeds a 18 d.p.a. (DS), ma u e seeds (MS), oo s and shoo s om 2–4-day- old seedlings, o lea es (L) om 7-week-old plan s. Molecula -mass ma ke s a e shown on he le -hand side. (C) P o ein ex ac s (15 µg) om scu ellum, aleu one laye o s a chy endospe m dissec ed om seeds a e 1–4 days o imbibi ion we e subjec ed o SDS/PAGE, elec o- ans e ed on o ni ocellulose shee s and p obed wi h whea an i-NTR an ibodies. The e o e his band may co espond o non-speci ic de ec ion by he an ibodies in ex ac s om he s a chy endospe m o he union o NTR wi h ano he p o ein in he p ocess o deg ada ion. The p esence o se e al NTR genes in plan s is no su p ising. In A abidopsis, a leas h ee di e en genes encode pu a i e NTRs (Figu e 1). One o hem, e e ed o as A NTRB in Figu e 1 (accession no. CAB54874), p e iously epo ed by Jacquo e al. [21], encodes o a p o ein o 35 kDa and shows a close simila i y (76%) o he whea NTR epo ed he e. A second one, e e ed o as A NTRA in Figu e 1 (accession no. AAB86519), encodes o a mi ochond ial NTR [8] and, he e o e, is p obably no de ec ed in ou Wes e n-blo analysis. Finally, a hi d gene encoding a pu a i e NTR, e e ed o as A NTRC in Figu e 1, was iden i ied in A abidopsis (accession no. AAB84351). As shown by he phylogene ic analysis, his gene shows a lowe le el o simila i y (52%) and encodes a p o ein wi h a deduced molecula mass o 58.3 kDa. I he monoco s con ain a simila NTR gene, ei he i is no exp essed in whea o i has no been de ec ed by he an ibody used in he p esen s udy. In e es ingly, he Wes e n-blo analysis o he accumula ion o NTR shows a wide dis ibu ion o his enzyme in mos whea issues as i occu s in hio edoxin h. Al hough i is ema kable ha he e is a low con en o NTR in seeds, whe e hio edoxins haccumula e a high le el [18], NTR accumula es in scu ellum and aleu one cells o seeds a e ge mina ion, in ag eemen wi h he inc ease in hio edoxins hin hese impo an issues o he ge mina ing seeds [18]. These esul s sugges an impo an ole o he NTR– hio edoxin hsys em o he success o ge mina ion. To da e, h ee hio edoxins hha e been desc ibed in whea #2002 Biochemical Socie y 497Thio edoxin educ ase om monoco s [18,29], bu i is likely ha mo e hio edoxins ha e p esen . To unde s and ully he ole o he NTR– hio edoxin hsys em in seeds o ce eals, i is necessa y o de e mine whe he hey show any speci ici y o exp ession in he di e en o gans and also o es he eac i i y o each o hese hio edoxins hwi h NTR. This wo k was suppo ed by g an no. BMC2001-2366 om Minis e io de Ciencia y Tecnologı!a, Spain and g an no. CVI-182 om Jun a de Andalucı!a, Spain. 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