Root tip contact with low-phosphate media reprograms plant root architecture

Transcription

1 27 Nture Pulishing Group Root tip ontt with low-phosphte mei reprogrms plnt root rhiteture Sergio Svistoonoff,2, urey Creff, Mtthieu Reymon,3,Céile Sigoillot-Clue, Lilin Riu, line lnhet,4, Lurent Nussume & Thierry Desnos Plnt roots re le to sense soil nutrient vilility. In orer to quire heterogeneously istriute wter n minerls 3, they optimize their root rhiteture. One poorly unerstoo plnt response to soil phosphte (P i ) efiieny is reution in primry root growth with n inrese in the numer n length of lterl roots 4 2. Here we show tht physil ontt of the riopsis thlin primry root tip with low-p i meium is neessry n suffiient to rrest root growth. We further show tht loss-of-funtion muttions in Low Phosphte Root (LPR) n its lose prlog LPR2 strongly reue this inhiition. LPR ws previously mppe s mjor quntittive trit lous (QTL) 2 ; the moleulr origin of this QTL is expline y the ifferentil lleli expression of LPR in the root p. These results provie strong eviene for the involvement of the root p in sensing nutrient efiieny, responing to it, or oth. LPR n LPR2 enoe multiopper oxises (MCOs), highlighting the essentil role of MCOs for plnt evelopment. To stuy the effet of minerl efiieny on root evelopment, we previously mppe LPR, QTL with lrge effet involve in primry root growth rrest in response to low P i n in the ontrol of primry root ell length 2,inn. thlin reominnt inre line (RIL) popultion erive from the (yreuth) n (hr) wil essions. From this RIL popultion we erive two ner isogeni lines (NIL) of line no. 94 (ref. 2). Seelings of the NIL 94, homozygous for the ominnt llele of LPR (LPR ), hve short root phenotype on low ( mm) P i ompre with the NIL 94 rrying the reessive LPR llele 2.Wehvefurthershown (Supplementry Fig. online) tht this root growth rrest is relte to low P i, s vrying two hemil prmeters in the growth meium tht ffet P i iovilility in soils 3 the ph n the Fe onentrtion h n effet on growth rrest (tht is, when the meium i not ontin Fe or h more si ph, the root growth ws not inhiite y low P i ). To etermine the moleulr sis of the LPR QTL, we ientifie the responsile gene LPR (tg23) y omining severl omplementry strtegies summrize here (see Methos for etils). First, y nlyzing the existing RIL olletion of the ross 4, we mppe the LPR QTL to 36-k region of hromosome I (Supplementry Tle online). Seon, in orer to generte mutnt lleles of the LPR QTL, we evise g-ry mutgenesis strtegy. Rition inues lrge eletions s well s point muttions.we therefore use pollen from g-ry mutgenize 94 plnts to pollinte flowers of 94 plnts n sreene for progeny (F) seelings with long primry root on low P i. In this wy, we isolte three point muttions in the LPR gene (in ition to 7 lrge eletion lleles of the LPR lous), eh hving strongly reue response to low P i (Supplementry Fig. 2 online n Fig. ). Two trnsfer DN (T-DN) insertion mutnts of tg23 (lpr- n lpr-2, Fig. ) generte in nother geneti kgroun (Col-) 6, whih re most proly null lleles (Supplementry Fig. 3, online), ehve similrly to the g-ry inue mutnts (Fig.,), wheres T-DN mutnts in the genes immeitely proximl or istl to tg23 h wil-type phenotype (t not shown). Thir, the lpr- T-DN llele i not genetilly omplement the LPR llele of the QTL (Supplementry Fig. 4 online). Fourth, moleulr onstrut ontining the LPR gene restore low-p i responsiveness to the inre line 94 (Fig. ). losely relte. thlin prlog of LPR, tg74 (herefter nme LPR2), hs 79% ientil mino is (t not shown). We isolte two T-DN insertion mutnts 6 of LPR2 (lpr2- n lpr2-2) tht re most proly null lleles (Supplementry Fig. 3,). nlysis of the lpr2 mutnts n of the lpr-,lpr2- oule mutnt showe tht LPR n LPR2 h similr n itive roles n were neessry for the root growth response to low P i (Fig. 2). The preite mino i sequenes of LPR n LPR2 re similr to those of MCOs 7. In prtiulr, they ontin the twelve opper ining mino is require for MCO tlyti tivity in, struturlly hrterize MCO of illus sutilis 8 (Fig. 3). Lortoire e iologie u Développement es Plntes, Déprtement Éophysiologie Végétle et e Miroiologie, Unité Mixte e Reherhe 69 Commissrit à l Énergie tomique (CE), Centre Ntionl e l Reherhe Sientifique (CNRS), Université ix-mrseille-ii, CE Crhe, 38 St. Pul-lez-Durne Ceex, Frne. 2 Present ress: Equipe Rhizogenese, Unité Mixte e Reherhe Diversité et pttion es Plntes Cultivées, Institut e Reherhe pour le Développement, 9, venue gropolis, Montpellier Ceex, Frne. 3 Present ress: Deprtment of Plnt reeing n Genetis, Mx Plnk Institute for Plnt reeing Reserh, Crl-von-Linné-Weg, D-829 Cologne, Germny. 4 Present ress: Monsnto, Route e Crest, 2674 Suzet, Frne. Corresponene shoul e resse to T.D. (thierry.esnos@e.fr). Reeive 2 Ferury; epte 2 pril; pulishe online 3 My 27; oi:.38/ng VOLUME 39 [ NUMER 6 [ JUNE 27 NTURE GENETICS

2 27 Nture Pulishing Group High-P i 94 Low-Pi LPR 94 TG lpr- oringly, we oserve tht in vitro, LPR oxiizes 2,2 zinois(3-ethylenzothizoline-6-sulfonte) (TS), wheres no MCO tivity ws etete with n LPR protein ontining the g3- muttion or mutte t one puttively ruil opper-ining histiine 9 (Fig. 3). If the MCO tivity of the LPR proteins is require for the low P i inue root growth rrest, then inhiiting this tivity shoul enhne wil-type root growth. To test this hypothesis, we grew wil-type (Col-) seelings on low-p i meium supplemente with either mm tetrthiomolyte (TTM) or mm soium fluorie (NF), two potent inhiitors of MCOs 2,2. t mm TTM, the wil-type primry root ws 2.8 times longer thn on the TTM-free ontrol meium (respetively 2.4 ±.6 mm n 4.4 ±.8 mm) n ws s long s tht of the lpr- mutnt (2.7 ±. mm) (Fig. 3). Similr results were otine with NF (Fig. 3). Thus, phenoopying the Lpr mutnt phenotype y treting wil-type with TTM or NF supports the view tht MCO tivity, most proly resulting from the LPR n LPR2 expression, is require for low P i epenent growth inhiition. In orer to fin the moleulr origin of the LPR QTL, we first ompre the protein sequenes of LPR n LPR. There re six mino i sustitutions, ut these re not in onserve MCO motifs (Fig. 3); n in RIL no. 98 reomintion in exon 2 of LPR (Supplementry Tle n Fig. 3) exlue the possiility tht the QTL is in the 3 hlf of the gene. Notly, the in vitro tivity of LPR ws not signifintly ifferent from tht of LPR (Fig. 3). Furthermore, the 94 line ws omplemente y trnsgene ontining the LPR oing sequene ple uner the ontrol of the promoter sequene of LPR (Supplementry Fig. online). lpr-2 TG * * * * * γ6-69 γ3- γ8-2 : CTCCGTTGTCCGGGCCCCT γ6-69: CTC--TGTCC----CCCCT : CC CCCCT γ3- γ8-2 γ6-69 Col- Ipr- Ipr-2 TT(I24) GT (S) Primry root length (mm) Deletion of T222 4 p stop 2 p ± ph.6 ph Col- 94 E E E E E E E D CD C CD Ipr- Ipr2- Ipr-, Ipr2-94 y γ3- γ8-2 γ6-69 Col- Ipr-2 Figure LPR is neessry for root growth inhiition y low P i.() Position of the T-DN insertions (tringles) n g-ry inue point muttions (rrows) in lpr lleles. *Copperining sites in LPR (see text n Fig. 3). () Phenotypes of 94 n 94 NILs, the g-ry inue 94 mutnts, Col- wil-type n the two insertion mutnts. Sle r, m. () Histogrm of the primry root length of the lpr mutnts grown for 9 on low-p i (white rs) or high-p i (lk rs) meium (men ± s.e.m., n ¼ 4 4 seelings). Vlues with iffering letters re signifintly ifferent t the P o. level. () Complementtion of NIL 94. Control lines (left pnel) n progeny of 94 plnt segregting for the LPR trnsgene (right pnel). Seelings were grown for 9 on low-p i meium. +, green fluoresene of seelings provie y the trnsformtion mrker GFP from the trnsgene; ±, fint fluoresene;, nonfluoresene. Tken together, these t imply tht the funtionl ifferene etween the LPR n LPR lleles is linke to the promoter sequenes of LPR (plpr) rther thn to the LPR enzymti tivity per se. When ompre with plpr, plpr h severl polymorphisms (sustitutions, insertions n eletions). However mny of these polymorphisms were shre with plpr Col- (t not shown), funtionl llele (s shown ove). Forty-one nuleoties upstrem of the LPR trnsription strt site, plpr h 6-p eletion (Fig. ). This smll prt of the promoter region must e ruil for LPR gene funtion, s the g-ry inue llele g6-69, erive from the LPR llele, ws mutte t this site (Fig. ) n ehve the sme s the g8 2 llele, whih is most proly null llele (Fig.,). These t imply tht, ompre with plpr, the funtion of 2 7 Col- 6 Ipr- Ipr2- Ipr-, Ipr µm P i ph.6 ph 6. Primry root length (mm) Figure 2 LPR n LPR2 hve similr n itive funtions. () Phenotype of Col- wil-type, lpr- n lpr2- single mutnts n lpr-,lpr2- oule mutnts. Seelings were grown for 9 on ph-.6 (top row) or ph-6. (ottom row) meium with P i t the onentrtions (mm) inite t the ottom. The genotypes of the four lines re s in the top left pnel. Sle r, m. () Histogrm of the primry root length of lines grown s in, men ± s.e.m., n ¼ 3 9 seelings. NTURE GENETICS VOLUME 39 [ NUMER 6 [ JUNE

3 27 Nture Pulishing Group Figure 3 LPR is multiopper oxise. () lignment of LPR n LPR protein sequenes with from illus sutilis. Re ots, mino is polymorphi etween LPR n LPR ; yellow line, region of the rossing over in RIL no. 98; lue rrowhes, positions of the g3- n g8-2 muttions; numers ove the sequenes, mino is ining the type, type 2 or type 3 opper toms in (ref. 8); *His68. Purple kgroun, mino is tht re ientil etween LPR proteins n ; green kgroun, mino is tht re similr. () MCO tivity in protein extrts from yest strins expressing LPR,LPR or the I24S or H48 mutnt forms of LPR. ove, immunolot n Coomssie gel of protein extrt from yest strins trnsforme with empty or LPR-expressing vetor (rrowhe, LPR). elow, LPR tlyti tivity; eh r, men (± s.e.m.) of triplite of four inepenent LPR yest lones minus the tivity of the empty-vetor ontrol. (,) Inhiitors of MCOs phenoopy the lpr mutnt phenotype. Effets of TTM () n NF () on the primry root length of Col- wil-type (white rs) n lpr- (lk rs) seelings grown on low-p i meium for 8 ys; men ± s.e.m., n ¼ 4 or seelings () or n ¼ 8 seelings (). Vlues with iffering letters re signifintly ifferent t the P o. level. plpr is somehow less effetive, possily ue to less trnsription. To test this hypothesis,wenlyzelpr mrn umultion in the two NILs 94 n 94 y quntittive RT-PCR (QRT- PCR). oth 94 n 94 seelings h LPR mrn in their roots n leves. However, LPR mrn unne in roots of 94 seelings ws 2. n 3-fol tht in 94, respetively in low n high P i (Fig. 4 n Supplementry Fig. 3). This is onsistent with the reessive nture of the LPR llele ompre with the LPR llele 2. The 94 seelings expresse LPR mrn enoing n tive MCO, n yet ehve s loss-of-funtion llele (Fig.,). In orer to unerstn this pprent prox, we exmine the expression pttern of LPR in more etil. We introue the trnsriptionl trnsgene -gluuronise (GUS) reporters plpr ::GUS n plpr ::GUS into the 94 kgroun. plpr ::GUS ws expresse in the root tip, inluing the meristemti region (where root ells re generte) n the root p (the smll group of ells wrppe roun the root tip) (Fig. 4). Notly, there ws less expression in the root tip of plpr ::GUS thn plpr ::GUS. In prtiulr, there ws little, if ny, expression of plpr ::GUS in the root p (Fig. 4, left pnel). We onfirme these results y semiquntittive RT-PCR performe on lser-miroissete root p tissues (Fig. 4,). These expression ptterns were onstitutive, s they were not linke to the P i,feorh + onentrtions in the growth meium (Supplementry Fig. 6 online) nor to the geneti kgrouns: in NIL 94 the two reporter onstruts gve root GUS stinings similr to tht in the 94 kgroun (Supplementry Fig. 6). ltogether, these t strongly inite tht the moleulr sis of the LPR QTL erives from the ifferent ptterns of LPR expression in the root tip. We teste two hypotheses tht oul explin low P i epenent root growth rrest: the first posits nutritionl response n the seon γ γ * posits signling response. oring to the first hypothesis, growth woul ese euse of internl phosphte efiieny in ells. To test this we mesure the P i ontent of roots. When grown on low P i for, the P i ontent of 94 n Col- wil-type roots ws not signifintly (P o.) ifferent from tht of 94 n lpr- (Supplementry Tle 2 online), initing tht the first hypothesis my e rejete. oring to the seon hypothesis, root growth rrest woul e triggere when the root tip senses the low P i onentrtion of the meium. This theory ws supporte y the result of omprtmente root-growth experiment, in whih we foun tht the primry root-growth rrest ourre when the root tip ws in ontt with the low-p i meium, even if leves were in ontt with high-p i meium (Fig. ). In nother experiment we oserve on low-p i pltes tht if primry root tip i not touh the gr meium the root growth ws not inhiite, ut if, in growing frther, the root tip eventully enountere the meium, then root growth soon ese (Supplementry Fig. 7 online). The rpi root growth rrest (less thn 2 ) fter trnsfer of Col- seelings from high- to low-p i mei (Supplementry Fig. ) is lso omptile with this seon hypothesis. Furthermore, this rrest orrelte with the rrest of root ells ivisions (Supplementry Fig. 6 n ref. ) n elongtion 2 n orroorte finings tht roots lolly sense n respon to low P i 22. Overll, these results strongly inite tht the root p is the site of the sensing n/or response to low onentrtions of exogenous P i. In summry, nlysis of. thlin nturl vrition llowe us to isolte the mjor QTL LPR ontrolling low P i triggere root growth inhiition. This QTL is expline y the ifferentil lleli expression of LPR in the root p, n orgn essentil for root meristem kd 7 7 LPR tivity (nkt liter ) Primry root length (mm) Primry root length (mm) I24S H68 Empty vetor I24S H68 C C µm TTM C D µm NF 794 VOLUME 39 [ NUMER 6 [ JUNE 27 NTURE GENETICS

4 27 Nture Pulishing Group LPR mrn reltive level. plpr ::GUS Pht;4 LPR R L R L mintenne 23 n uxin fluxes 24. We propose tht when the primry root tip rehes low-p i zone, the LPR proteins of the root p moify the tivity n/or istriution of hormone-like ompoun. This triggers the primry root evelopmentl swith from ineterminte to eterminte growth, the reution of ell elongtion n the promotion of lterl roots. This is the first emonstrtion tht MCOs hve role in plnt evelopment in response to n ioti signl. s oth prokryotes n eukryotes hror MCOs 2, these finings my ontriute to unerstning other evelopmentl proesses. METHODS Plnt mteril n growth onitions. The SLK lines 6 were provie y the Nottinghm riopsis Stok Centre. For the QTL fine mpping we use the 4-RIL popultion previously esrie 4 (see vnt/ for etils). Seeling n plnt growth onitions were s previously esrie 2.The94 n 94 lines re NILs with, respetively, or llele in the MST. ng248 region 2. Unless otherwise inite, the growth meium ws uffere t ph.6 with 3. mm 2-(N-morpholino)ethne sulfoni i (MES) uffer efore utolving. The mmonium tetrthiomolyte ws from lrih n NF from Prolo. Fine mpping of LPR. We sreene the popultion of 4 RILs for lines rrying reominnt hromosome in the 2.6-M intervl flnke y the plpr ::GUS Figure 4 In the root tip, less LPR thn LPR is expresse. () Histogrm of the QRT-PCR nlysis of LPR mrn in leves (L) n roots (R) of NIL 94 n 94 seelings grown on high P i (+) or low P i ( ). Pht;4 is low P i inue ontrol gene 3. Men (± s.e.m.) of triplite of three inepenent QRT-PCR retions. Dt normlize to 94 roots in high P i.() GUS stining of the root tips of 94 seelings rrying the plpr ::GUS (left) or the plpr ::GUS (right) onstrut. Sle r, mm. (,) LPR mrn unne in root p. () Root tip efore (left) n fter (right) lser miroissetion of the root p. Sle r, mm. () Semiquntittive RT-PCR nlysis of LPR mrn unne in the miroissete root ps of 94 n 94 seelings. Dt normlize to 94 ; men ± s.e.m. (n ¼ 3). LPR mrn reltive level +3 moleulr mrkers MST. n ng248. We selete 48 RILs n phenotype them in low-p i onitions, n fine-mppe the reomintion rekpoints with newly evelope mirostellite mrkers (Supplementry Tles n 3 online), llowing us to lolize LPR to 6-k intervl. We then nrrowe LPR own to 38-k intervl y sequening DN of two RILs with reomintion rekpoints in lose proximity to LPR (Supplementry Tle ). See Supplementry Methos online for further etils. Gmm-ry mutgenesis n ientifition of the lpr -mutnts. Eight flowering 94 /94 plnts were expose to 2 Gy (7 Gy min )ofg-rys from 6 Co soure. We use the irrite pollen to mnully pollinte the strte flowers of nineteen 94 /94 plnts, n sowe, resulting F sees on low-p i pltes. In the puttive lpr F mutnts, we mppe the g-ry inue eletions with PCR mrkers lote etween MST. n ng248 (Supplementry Fig. 2). In four F plnts we i not etet lrge eletions; in their F2 progeny we selete seelings homozygous for the llele using PCR mrkers in the LPR region n sequene the tg23 gene. The sequene of eh point muttion ws verifie in seon mutnt siling. See Supplementry Methos for further etils. Moleulr onstruts. For omplementtion, we PCR-mplifie from genomi DN the tg23 gene of the ession, inluing 2. k upstrem of the TG n 24 p ownstrem of the stop oon, n lone it into the pfp vetor ( yieling the LPR -pfp onstrut. For the promoter-gus fusion, we mplifie from genomi DN 2.-k frgment upstrem of the TG of the tg23 gene from the 94 n 94 lines n lone it in pxcsg-gfp erive vetor 26 (L. Noël, CE Crhe, unpulishe t) in whih the GFP gene ws reple y the GUS gene, yieling the plpr ::GUS n plpr ::GUS onstruts, respetively. These ifferent onstruts were introue 27 into the 94 n 94 lines n trnsformnts were selete either uner UV light for the LPR -pfp onstrut or y ST (grevo) seletion in soil for the other onstruts. See Supplementry Methos n Supplementry Tle 3 for further etils. Rel-time QRT-PCR. We rrie out rel-time QRT-PCR using n I 7 (pplie iosystems) with SYR Premix ExTq (Perfet Rel Time) s in ref. 28. Stnr urves were generte y seril ilutions of first-strn DN preprtions. Lser miroissetion of root ps n semiquntittive RT-PCR. We grew 94 n 94 seelings 7 on high-p i meium n then ut their primry roots n iretly eposite the roots on the plsti film of lser miroissetion slies (Lei). We performe the miroissetions uner High-P i High-P i Low-P i Low-P i Low-P i High-P i High-P i Low-P i Figure Root growth on low-p i meium is inhiite through the root tip. Col- wil-type seelings first grown for 3 on high-p i meium (not shown here) n then trnsferre t y ( ) to the inite omprtmentlize vertil pltes suh tht the upper n lower prts of eh seeling were in ontt with ifferent mei. () Upper prt of the seelings on high-p i meium, lower prt on low-p i or high-p i meium. () Upper prt of the seelings on low-p i meium, lower prt on high-p i or low-p i meium. Lower pnels, the sme pltes 3 lter ( +3 ). Sle r, 2. mm. NTURE GENETICS VOLUME 39 [ NUMER 6 [ JUNE 27 79

5 27 Nture Pulishing Group LMD6 (Lei) mirosope. For eh line, 8 root ps were ollete in 7 ml of RN extrtion uffer ontining mm DTT; tues were then store t 8 C. Totl RN ws extrte with the RNesy Miro Kit () (Qigen) oring to the mnufturer s instrutions n elute in 4 ml RNse-free wter. First-strn DN synthesis is esrie in the Supplementry Methos. Semiquntittive RT-PCR ws performe on n ep-grient-s thermoyler (Eppenorf) n the reltive expression of LPR mrn ws normlize to the mount of the root p speifi CEL mrn 29. GUS stining. We selete lines whih gve 3: segregtion of knmyin resistne (rrie y the T-DN) n grew them for 9 on high-p i meium. GUS stining of plnt tissues ws performe s previously esrie 29 exept tht seelings were inute in the stining solution for 6 h n then trete with 7% ethnol t C for h. GUS stining ws repete four times eh with two inepenent lines for eh onstrut, n ¼ seelings per line. These four experiments gve similr results. LPR MCO tivity. Wil-type n mutnt LPR proteins were proue in Shromyes erevisie n the MCO tivity ssye on TS with totl protein extrts (Supplementry Methos). Immunolotting. Yest protein extrts were seprte y SDS-PGE n lotte. The memrne ws soke with polylonl ntioy to (see knowlegments) n stine with n lkline phosphtse onjugte got ntioy to rit IgG (Sigm). ession oes. Gennk: LPR oing sequene, DQ66363; LPR oing sequene, DQ PD:, GSK. riopsis thlin:, N7923;, N7924; Columi (Col-), CS6; lpr-, SLK_6297; lpr-2, SLK_267; lpr2-, SLK_993; lpr2-2, SLK_6362. Note: Supplementry informtion is ville on the Nture Genetis wesite. CKNOWLEDGMENTS We grtefully thnk the Nottinghm riopsis Stok Centre for proviing SLK insertion lines; J. Viente for the g-ry irrition; L. Mrtins (Instituto e Tenologi Quími e iológi, Oeirs) for the ntioy; L. Noël (CE Crhe) for the pxcsg-gfp vetor; F. Pry (CNRS, Grenole) for the pfp vetor; T. Tron (CNRS, Mrseille) for the yest vetors; N. Leonhrt for helping with QRT-PCR; C. Sllu for the 96-well DN extrtion protool;. Croff for helping with lser miroissetion; C. Srroert for her erly involvement on Fe stuies; the Groupement e Reherhes ppliquées en Phytotehnologie tem for tking re of plnts; J.-L. Montillet for helpful isussions; n M. Crespi, M. Koornneef, G. Kunze, E. Mrin-Nussume, L. Noël n M.-C. Thiu for ritil reing n suggestions on n erly version of the mnusript. We thnk R. Crol for orreting the English on n erly version of the mnusript. S.S. ws supporte y the Frenh Ministère e l Reherhe n y the CE; C.S.-C., M.R. n L.R. were supporte y the CE. UTHOR CONTRIUTIONS S.S.,.C. n T.D. performe most of the experiments. M.R. ontriute to the fine mpping of LPR n performe sttistil nlysis; C.S.-C. performe enzymti nlysis of LPR; L.R. omplemente the 94 line;.. isolte the lpr2 mutnts. S.S. n T.D. nlyze the t n wrote the mnusript with input from L.N. T.D. oneive n supervise the projet. COMPETING INTERESTS STTEMENT The uthors elre no ompeting finnil interests. 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Genome-wie trnsriptome profiling of the erly mium response of riopsis roots n shoots. iohimie 88, 7 76 (26). 29. el Cmpillo, E., el-ziz,., Crwfor, D. & Ptterson, S.E. Root p speifi expression of n eno--,4-d-glunse (ellulse): new mrker to stuy root evelopment in riopsis. Plnt Mol. iol. 6, (24). 3. Misson, J., Thiu, M.-T., ehtol, N., Rghothm, K. & Nussume, L. Trnsriptionl regultion n funtionl properties of riopsis Pht;4, high ffinity trnsporter ontriuting gretly to phosphte uptke in phosphte eprive plnts. Plnt Mol. iol., (24). 796 VOLUME 39 [ NUMER 6 [ JUNE 27 NTURE GENETICS