Beta-Cyanoalanine synthase action in root hair elongation is exerted at early steps of the root hair elongation pathway and is independent of direct cyanide inactivation of NADPH oxidase

Running head: Cyanide ac ion in oo hai elonga ion Co esponding au ho : D . I. Ga cía. Ins i u o de Bioquímica Vege al y Fo osín esis. Consejo Supe io de In es igaciones Cien í icas and Uni e sidad de Se illa. A enida Amé ico Vespucio, 49, 41092 Se illa, Spain. Tel: + 34 954 489 500. Fax: +34 954 460 165. Email: i ene.ga cia@ib .csic.es Subjec a ea: g ow h and de elopmen This manusc ip con ains 4 black and whi e igu es, 3 colo igu es, 2 ables, 7 supplemen a y igu es and 1 supplemen a y ideo. © The Au ho 2018. Published by Ox o d Uni e si y P ess on behal o Japanese Socie y o Plan Physiologis s. All igh s ese ed. Fo Pe missions, please e-mail: jou nals.pe [email protected] Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 2 ß-Cyanoalanine syn hase ac ion in oo hai elonga ion is exe ed a ea ly s eps o he oo hai elonga ion pa hway and is independen on di ec cyanide inac i a ion o NADPH oxidase Running head: Cyanide ac ion in oo hai elonga ion Lucía A enas-Al onseca, Cecilia Go o , Luis C. Rome o and I ene Ga cía* Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones Cien í icas and Uni e sidad de Se illa, A enida Amé ico Vespucio, 49, 41092 Se illa, Spain * Email: i ene.ga cia@ib .csic.es. Fax: +34 954 460 165 Abb e ia ions ACC: 1-aminocyclop opane-1-ca boxylic acid bHLH: basic helix-loop-helix CAS: ß-cyanoalanine syn hase COB: 2-hyd oxocobalamin CT: cycle h eshold FW: esh weigh GFP: g een luo escen p o ein H 2 DCFDA: 2',7'-dichlo odihyd o luo escein diace a e NBT: ni o blue e azolium ORF: open eading ame ROS: eac i e oxygen species TF: ansc ip ion ac o XTT: 3′-[1-[(phenylamino)-ca bony]-3,4- e azolium]-bis(4-me hoxy-6-ni o)benzene-sul onic acid Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 3 Abs ac In A abidopsis haliana, cyanide is p oduced concomi an ly wi h e hylene biosyn hesis and is mainly de oxi ied by he ß-cyanoalanine syn hase CAS-C1. In oo s, CAS-C1 ac i i y is essen ial o main ain a low le el o cyanide o p ope oo hai de elopmen . Roo hai elonga ion elies on pola ized cell expansion a he g owing ip, and we ha e obse ed ha CAS-C1 loca es in mi ochond ia and accumula es in oo hai ips du ing oo hai elonga ion, as shown by obse ing he luo escence in plan s ans o med wi h he ansla ional cons uc P oC1:CASC1-GFP, con aining he comple e CAS-C1 gene used o GFP. Mu an s in he SUPERCENTIPEDE (SCN1) gene, ha egula e he NADPH oxidase RHD2/A bohC, a e a ec ed a he e y ea ly s eps o he de elopmen o oo hai ha do no elonga e and do no show a p e e en ial localiza ion o he GFP accumula ion in he ips o he oo hai p imo dia. Roo hai s o mu an s in CAS-C1 o RHD2/A bohC, which ca alyzes he gene a ion o ROS and he Ca 2+ g adien , co ec ly s a o g ow ou bu hey do no elonga e ei he . Gene ic c osses be ween he cas-c1 mu an and scn1 o hd2 mu an s we e pe o med and he de ail pheno ypic and molecula cha ac e iza ion o he double mu an s demons a e ha scn1 mu a ion is epis a ic o cas-c1 and cas-c1 is epis a ic o hd2 mu a ion, indica ing ha CAS-C1 ac s in ea ly s eps o he oo hai de elopmen p ocess. Mo eo e , ou esul s show ha he ole o CAS-C1 in oo hai elonga ion is independen o H 2 O 2 p oduc ion and o a di ec NADPH oxidase inhibi ion by cyanide. Keywo ds A abidopsis haliana, ß-Cyanoalanine Syn hase, Roo Hai , Cyanide, SCN1, RHD2. Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 4 In oduc ion Cyanide is ex ensi ely p esen in all o ganisms, including bac e ia, ungi, insec s and plan s. Howe e , cyanide is a oxic compound due o i s high eac i i y wi h ke o compounds and Schi bases and because i chela es di- and i alen me al ions in he p os he ic g oups o me allop o eins (Dona o e al. 2007). Mi ochond ia is he majo a ge o cyanide, whe e i binds o he heme i on o cy och ome c oxidase, blocking he espi a o y chain (Coope and B own 2008). In non-cyanogenic plan species such as A. haliana, he main sou ce o cyanide is he biosyn hesis o he ho mone e hylene, which is in ol ed in egula ing nume ous de elopmen al p ocesses and esponses o s ess condi ions (Bleecke and Kende 2000), and he biosyn hesis o he phy oalexin camalexin, which is o med when A. haliana plan s a e in ec ed by a la ge a ie y o mic oo ganisms (Glawischnig 2007). The e o e, unde ce ain de elopmen al o en i onmen al condi ions, plan s p oduce signi ican amoun s o cyanide ha may be ha m ul o hei cells, equi ing de oxi ica ion. To keep cyanide below oxic concen a ions, plan s possess di e en me abolic pa hways, he main pa hway o which is ha in ol ing ß-cyanoalanine syn hase (CAS) ((Machingu a e al. 2016) and e e ences he ein). A abidopsis plan s exp ess he mi ochond ial ß-cyanoalanine syn hase CAS-C1 ( o me ly CYS-C1, (Rome o e al. 2014) (Wa anabe e al. 2008)), which, oge he wi h he O-ace ylse ine( hiol)lyases (OASTLs), belongs o he amily o ß-subs i u ed alanine syn hase enzymes. CAS is a py idoxal phospha e-dependen enzyme ha uses cys eine o de oxi y cyanide by con e ing cyanide and cys eine in hyd ogen sul ide (H 2 S) and ß-cyanoalanine. H 2 S also blocks he mi ochond ial espi a o y pa hway and he e o e needs o be de oxi ied by he ue OASTL OAS-C, which inco po a es H 2 S o O-ace ylse ine (OAS) o p oduce cys eine, which is ecycled by CAS-C1 o de oxi y cyanide, hus comple ing he cyanide de oxi ica ion cycle in mi ochond ia (Al a ez e al. 2012). ß- Cyanoalanine is con e ed o Asn, Asp and ammonia by NIT4 class ni ilases, he eby ecycling he ni ogen o plan u iliza ion (Pio owski 2008). Cyanide a non- oxic le els has been sugges ed o pe o m egula o y oles in di e en physiological p ocesses. In animal sys ems, o example, i has been hypo hesized o ac as a neu omodula o (Cipollone and Visca 2007). In plan s, exogenously applied cyanide can ac as a egula o o seed do mancy and ge mina ion (Be hke e al. 2006; Chi asa and Ca 1998; Cohn and Hughes 1986; Seo e al. 2011; Siegien and Boga ek 2006; Wong e al. 2002) and play a ole in esis ance o i al and ungal pa hogens (Be hke e al. 2006; Chi asa and Ca 1998; Cohn and Hughes 1986; Seo e al. 2011; Siegien and Boga ek 2006; Wong e al. 2002). Ou p e ious in es iga ion on he CAS-C1 null mu an has p o ided insigh in o he ole o he endogenously p oduced Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 5 cyanide in A abidopsis. Loss-o - unc ion mu a ion o he CAS-C1 gene leads o a non- oxic inc ease in cyanide (Ga cia e al. 2010) and an al e ed immune esponse, i.e., inc eased suscep ibili y o he nec o ophic ungus Bo y is cine ea and inc eased ole ance o he bio ophic pa hogens Pseudomonas sy ingae p . oma o DC3000 and bee cu ly op i us (Ga cia e al. 2013). Because he null mu an exhibi s an induced al e na i e oxidase espi a ion, eac i e oxygen species (ROS) accumula ion and salicylic acid-dependen pa hway induc ion, i was hypo hesized ha cyanide migh gene a e a mi ochond ial signal, unknown o da e, ha could modula e he plan immune sys em (Ga cia e al. 2014). The cas-c1 mu an also exhibi s a oo hai less pheno ype, which is e e ed ei he gene ically by complemen a ion wi h he co esponding CAS-C1 gene o chemically by he addi ion o he cyanide an ido e hyd oxocobalamin (Al a ez e al. 2012; Ga cia e al. 2010). T ansc ip ional p o iling o he cas- c1 mu an e eals ha he genes encoding enzymes in ol ed in cell wall ebuilding and oo hai o ma ion a e unde exp essed in he mu an , as a e ce ain genes in ol ed in e hylene signaling and me abolism (Ga cia e al. 2010). Roo hai s a e ubula ex ensions o oo epide mal cells p oduced in he di e en ia ion zone o he oo and con e he abili y o abso b nu ien s and wa e , in e ac wi h mic obes, and physically ancho he plan o he soil. Due o he biological impo ance o hese s uc u es, which a e also a model o s udying ip g ow h in plan s, he molecula mechanisms in ol ed in he speci ica ion, di e en ia ion, and physiology o oo hai s in A abidopsis ha e been ex ensi ely e iewed (Ca ol and Dolan 2002; G ie son e al. 2014; Ishida e al. 2008). Roo hai speci ica ion is de e mined by posi ion-dependen signaling and molecula eedback loops. Once an ini ia ion si e has been selec ed, cell pola i y is es ablished, and a small swelling o ms. Roo hai elonga ion elies on pola ized cell expansion a he g owing ip, which in ol es mul iple in eg a ed p ocesses, including cell sec e ion, endomemb ane a icking, cy oskele al o ganiza ion, and cell wall modi ica ions. Sus ained oo ip g ow h in ol es oscilla ions in ex acellula pH, ROS and cy osolic calcium (Monshausen e al. 2007). Elonga ion is accompanied by gene a ion o a ip-high calcium g adien ha can be obse ed h oughou he emainde o oo hai g ow h (Dolan e al. 1994; Samaj e al. 2004; Schie elbein e al. 1992; Wyme e al. 1997). Thus, ROS a e equi ed o he ac i a ion o calcium channels and calcium ac i a es NADPH oxidase in a sys em o posi i e eedback ha main ains cell pola i y du ing oo hai elonga ion (Gappe and Dolan 2006; Takeda e al. 2008). The genes in ol ed in each o he phases o oo hai de elopmen ha e been iden i ied, including ansc ip ion ac o s, cell wall-modi ying enzymes, p o ein om he sec e ion appa a us, and p o eins ela ed o Ca 2+ p oduc ion, ROS gene a ion and managemen , cy oskele on, e c. ( e iewed in (Ca ol and Dolan 2002; G ie son and Schie elbein 2009)). Ho mones also play a ole in oo hai de elopmen , p incipally auxin Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 6 and e hylene ((G ie son e al. 2014) and e e ences he ein). The goal o his wo k is o u he ou unde s anding o he ole o CAS-C1 and cyanide in oo hai de elopmen . To his end, a cellula s a egy was implemen ed o obse e he spa io empo al exp ession and subcellula localiza ion o CAS-C1, wi h a pa icula ocus on de eloping oo hai s. Mu a ions in he SUPERCENTIPEDE1 (SCN1) gene a ec e y ea ly s eps o he oo hai de elopmen , esul ing in oo hai bulges ha do no elonga e. The SCN1 p o ein is a Rho GTPase GDP dissocia ion inhibi o ha unc ions as a nega i e egula o o Rho- ela ed plan GTPases (ROPs). Th oughou hai de elopmen , ROP GTPases ROP2, ROP4 and ROP6 localize o he ea lies swelling o he basal egion and ip. ROPs egula e he ac i i y o he NADPH oxidase encoded by ROOT HAIR DEFECTIVE 2 (RHD2)/A bohC, which ca alyzes he gene a ion o ROS and pa icipa es in he gene a ion o he Ca 2+ g adien a he ip o he oo hai ha d i es oo hai elonga ion (Fo eman e al. 2003; Ishida e al. 2008; Jones e al. 2007). Indeed, SCN1 ac s on he oo hai ini ia ion, swelling o ma ion, ansi ion o ip g ow h and ip g ow h s eps o oo hai o ma ion (Fo eman and Dolan 2001; Pa ke e al. 2000). In scn1 mu an s, ROP2 is mislocalized, and supe nume a y hai ini ia ion si es a e o med ha do no elonga e (Ca ol e al. 2005). Mu an s on hd2, on his pa , a e no able o p oduce supe oxide anion and show abno mal sho oo hai s (Fo eman e al. 2003). To demons a e epis a ic ela ionships in he oo hai elonga ion p ocess, gene ic c osses be ween he cas-c1 mu an and scn1 o hd2 mu an s ha e been pe o med, and some clues ega ding he unde lying mechanisms a e discussed. Resul s CAS-C1 localizes o mi ochond ia and accumula es in oo hai ips Based on i s sequence and he capaci y o he N- e minal po ion o he p o ein o di ec he g een luo escen p o ein (GFP) o mi ochond ia in ansien exp ession expe imen s (Yamaguchi e al. 2000), CAS-C1 has been classi ied as a mi ochond ial p o ein. Aiming o be e un a el he ole o CAS-C1 in ela ion o i s spa io- empo al exp ession, we cons uc ed a p omo e -genomic open eading ame (ORF)-GFP cons uc and ans o med plan s as desc ibed in he Ma e ials and Me hods sec ion and schema ized in Supplemen a y Fig. S1. We assumed ha he in e genic PIP1-CAS-C1 egion should con ain all ansc ip ional egula o y signals o Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 7 CAS-C1 exp ession and ha he genomic CAS-C1 egion, om +1 o he STOP codon, should con ain he egula o y sequences o he subcellula loca ion o he p o ein as well as possible addi ional ansc ip ional egula o y elemen s (Gu ie ez-Alcala e al. 2005). A ansla ional usion was hen ob ained by joining 2676 bp o he genomic sequence con aining 1442 bp o he comple e in e genic PIP1-CAS-C1 egion plus 1234 bp om he CAS-C1 ATG o he GAT be o e he STOP codon (Supplemen a y Fig. S1, www.a abidopsis.o g), o he GFP gene ca ied on he pMDC110 Ga eway ec o . The plan ans o ma ion cons uc was named P oC1:CASC1-GFP. T ansgenic plan s we e ob ained in di e en backg ounds, he wild ype (8 independen lines) and he cas-c1 (3 independen lines), scn1-1 (2 independen lines) and hd2GK (6 independen lines) mu an s, and analyzed by con ocal mic oscopy o in i o GFP de ec ion. No ele an di e ences o in ensi y o localiza ion o he luo escence we e obse ed among he di e en lines in any case, so one T3 line o each geno ype was selec ed o u he s udies. The spa io- empo al exp ession o CAS-C1 was examined by obse ing he luo escence in di e en issues o wild ype A abidopsis plan s ans o med wi h he P oC1:CASC1-GFP cons uc . Roo issues p esen ed a homogeneous do ed pa e n o luo escence in wild ype backg ound (Fig. 1A, D, G), which was consis en wi h a mi ochond ial localiza ion o he usion p o ein. This was con i med by co-localiza ion o GFP luo escence wi h he mi ochond ion-speci ic dye Mi o acke Deep Red 633 (Supplemen a y Fig. S2). In he oo , he signal was e y in ense, especially in he me is ema ic zone (Fig. 1A, B, C), likely due o he high mi ochond ia concen a ion in cells ha a e me abolically ac i e. In he oo hai , a s ong luo escence a he ip was obse ed in wild ype backg ound (Fig. 1D, G), ollowing he apical g ow h cha ac e is ic o oo hai s (Supplemen a y Video S1). This apical localiza ion o he luo escence was de ec ed om he e y beginning o oo hai de elopmen , a he swelling egion in he ini ia ion s age o he ip o he well- o med oo hai (Supplemen a y Video S1). These esul s we e consis en wi h he p oposed ole o CAS-C1 in oo hai o ma ion by modula ing he accumula ion o cyanide p esen in he g owing ip (Ga cia e al. 2010), which would ac as a ep esso o his p ocess om he ini ial s eps. In e es ingly, ans o ma ion o cas-c1 mu an plan s wi h he P oC1:CASC1- GFP cons uc e e ed, al hough pa ially, he oo hai less pheno ype (Supplemen a y Fig. S3), showing he complemen ed line accumula ion o GFP in he oo hai ip as in wild ype backg ound (Supplemen a y Fig. S3 B, C). Fu he mo e, we obse ed ha in he scn1-1 mu an , despi e being unable o p oduce oo hai p imo dia, he P oC1:CASC1-d i en GFP luo escence was localized in he mi ochond ia, ye in oo hai s i did no show a p e e en ial localiza ion o accumula ion a he ips o he oo hai p imo dia (Fig. 1B, E, H). The e o e, he Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 8 scn1-1 mu a ion does no a ec he subcellula localiza ion o CAS-C1 bu a he o he pola ized loca ion o mi ochond ia a he ip o he de eloping hai . Simila ly, in a T-DNA inse ion oo hai -speci ic NADPH oxidase hd2 mu an , hd2GK, he luo escence was localized a he mi ochond ia as well, al hough i did no show a oo hai ip speci ic localiza ion (Fig. 1C, F, I). The e o e, ou da a we e consis en wi h p e ious wo k showing ha mi ochond ia a e p esen a a high densi y in ip-g owing cells and spa ially associa ed wi h he ini ia ion and elonga ion o he oo hai bulge (Ca ol and Dolan 2002; Wang e al. 2010). Since cyanide is a po en inhibi o o he cy och ome espi a ion pa hway and oo hai elonga ion is a apid cell expansion p ocess wi h high cos o ene gy, we analyzed whe he he inc ease in cyanide in cas-c1 mu an s could ha e a de imen al e ec because o his p esumed inhibi ion o he ene gy sou ce equi ed o apid ip g ow h. P e ious epo has shown ha nei he he espi a ion a e is diminished, no he localiza ion o mi ochond ia is al e ed wi hin he oo hai in he cas-c1 mu an compa ed o wild ype (Ga cía e al 2010). Howe e , we ha e u he analyzed he le els o he main ene gy sou ce, ATP, in wild ype and cas-c1 mu an s in o de o ensu e ha he ene gy sou ce is no lacking in he cas-c1 oo s. Table 1 shows ha ATP le els we e indis inguishable in wild ype and cas-c1 mu an s, hus uling ou a dele e ious e ec o cyanide on he p oduc ion o he ene gy sou ce equi ed o oo hai g ow h. This esul leads us o deepen he concep o cyanide and/o CAS as an independen signaling componen in oo hai de elopmen . cas-c1 mu a ion is hypos a ic o he scn1-1 and epis a ic o he hd2-1 mo phological pheno ypes As CAS-C1 appea s o be in ol ed in he egula ion o he oo hai g ow h, we u he examined he exis ence o a ela ionship be ween CAS-C1 and he oo hai elonga ion pa hway. To es ablish he gene ic epis asis be ween hem, gene ic c osses we e ca ied ou be ween scn1-1 (Pa ke e al. 2000) o hd2-1 (Fo eman e al. 2003) and cas-c1 mu an s. Double scn1-1 cas-c1 and hd2-1 cas-c1 mu an s we e gene a ed and con i med by sequence and PCR analyses (Supplemen a y Fig. S4 and S5). Roo pic u es o wild ype, pa en al mu an s and he double scn1-1 cas-c1 and hd2-1 cas-c1 mu an s a 6-7 d a e sowing we e cap u ed unde a mic oscope (Fig. 2). The oo hai s o he cas-c1 mu an co ec ly began o g ow and de eloped small bulges (Fig. 2B, F), and scn1-1 displayed mul iple si es o hai g ow h eme ging om hai - o ming cells (Fig. 2C) and hd2-1 showed sho mal o med oo hai s (Fig. 2G). The double scn1-1 cas-c1 mu an exhibi ed a pheno ype ha was indis inguishable om ha o he scn1-1 simple mu an , showing mul iple oo hai p imo dia ha did no elonga e (Fig. 2D), whe eas he double hd2-1 cas-c1 mu an exhibi ed a pheno ype iden ical o he cas-c1 Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 9 simple mu an , i.e., small p o ube ances a he han sho oo hai s, mo e spa se han in he hd2-1 mu an (Fig. 2H). This e eals a ole o CAS-C1 in an in e media e poin be ween scn1 and he NADPH oxidase ac ion in he oo hai elonga ion p ocess. Addi ion o he e hylene dono ACC o he cul u e medium did no elonga e oo hai cells in any o he single cas-c1 o double hd2-1 cas-c1 o scn1-1 cas-c1 mu an s, demons a ing he independence o cas-c1 mu a ion and e hylene p oduc ion (Supplemen a y Fig. S6), (Ga cia e al. 2010). In addi ion, we ook ad an age o he use o he 2-hyd oxocobalamin (COB) compound as an an ido e o cyanide poisoning because i eac s wi h ee cyanide, p oducing cyanocobalamin, o i amin B12, which is no oxic (Bo on e al., 2007; Hall e al., 2007). Addi ion o COB o he cul u e medium has no e ec on he wild- ype oo bu is able o pa ially complemen he loss-o -hai pheno ype o he cas-c1 mu an (Ga cia e al. 2010). By compa ing he pheno ypes o COB- ea ed plan s, we ound ha cas-c1 could pa ially eco e he de elopmen o oo hai s, as p e iously epo ed, bu ha nei he he scn1-1 no he double scn1-1 cas-c1 mu an s showed e en pa ial e e sion o hei loss o he oo hai elonga ion pheno ype (Fig. 3C e sus 3G, 3D e sus 3H). These indings sugges ha cyanide accumula ion as he esul o cas-c1 loss-o - unc ion is no in ol ed in he scn1-1 pheno ype. On he o he hand, COB ea men was able o e e he hai less oo pheno ype o he double mu an hd2-1 cas-c1 o he single hd2-1 mu an (Fig. 3L e sus 3P), bu COB did no e e he hd2-1 mu an pheno ype (Fig. 3K e sus 3O) unde he condi ions es ed. The abo e obse a ions we e suppo ed by u he molecula da a. Cyanide accumula ion in he cas-c1 mu an has been desc ibed o ep ess se e al genes encoding enzymes in ol ed in he o ma ion o he oo hai ip, mainly cell wall- ela ed p o eins including FLA6 and MRH5, an a abinogalac an p o ein and a glyce ophospho yl dies e phosphodies e ase-like GPI-ancho ed p o ein espec i ely (Ga cia e al. 2010). Roo hai g ow h includes a a ie y o cellula componen s and compounds ha wo k in conce (Ca ol and Dolan 2002); Mend inna and Pe sson 2015). The basic helix-loop-helix (bHLH) ansc ip ion ac o (TF) ROOT HAIR DEFECTIVE 6 RHD6 con ols he ini ia ion o he oo hai (G ie son e al. 2014) whe eas he bHLH TF RHD6-LIKE 4 RSL4 is a di ec ansc ip ional a ge o RHD6, and i has been desc ibed o in eg a e he de elopmen al p og am aiming o egula e he pola g ow h (Ma zol e al., 2017, Vijayakuma e al., 2016). In o de o deepen in he epis a ic ela ionships be ween cas-c1 and scn1 o hd2, eal- ime quan i a i e RT-PCR was conduc ed. Figu e 4 shows ha all genes analyzed we e down- egula ed in cas-c1, scn1-1 and hd2-1 mu an s when compa ed o wild ype. When compa ing he double scn1-1 cas-c1 mu an wi h he single scn1-1 mu an , we could app ecia e ha he e we e no u he ep ession o RHD6, RSL4 o MHR5 and only a sligh ep ession o FLA6 (Fig. 4A). This sugges s ha he cas-c1 mu a ion did no signi ican ly a ec he beha io o Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 16 Roo issues we e incuba ed wi h a 20 nM solu ion o Mi oT acke Deep Red (Molecula P obes) o 10 min a oom empe a u e. Samples we e obse ed using a Leica HCX PLAN-APO 363 1.4 NA oil imme sion objec i e wi h a Leica TCS SP2 spec al con ocal mic oscope (Leica Mic osys ems). The dye was exci ed using a helium- neon lase a 644 nm, ei he in single con ocal op ical sec ions o in se ial op ical sec ions. Emi ed ligh was collec ed h ough a iple dich oic beam spli e (TD 488/543/633) and de ec ed a e spec al sepa a ion in he 650 o 700 nm ange (pseudocolo ed blue). De ec ion o ROS Fo de ec ion o he supe oxide anion, oo s we e s ained wi h NBT (Ni o e azolium blue chlo ide, Sigma- Ald ich) as desc ibed p e iously (Ga cia e al. 2010). Seedlings we e incuba ed in 0.1 M T is-HCl, 0.1 M NaCl, 0.05 M MgCl 2 , and 0.5 mg mL -1 NBT (pH 9.5) o 2 h a oom empe a u e in he da k. A e insing, oo s we e imaged unde b igh - ield illumina ion unde an Olympus BX50 mic oscope and images we e aken using a Leica DFC300FX digi al came a. Fo luo ime ic de ec ion o H 2 O 2 , oo s we e incuba ed o 5 min wi h 10 mM H 2 DCFDA (Li e Technologies) in he p esence o 10 mM p opidium iodide (Li e Technologies) o isualize cell walls. The samples we e obse ed using a TCS SP2 spec al con ocal mic oscope (Leica Mic osys ems) wi h he ollowing se ings: exci a ion, 488 nm; emission, 500 o 550 nm o luo escein de ec ion and 600 o 650 nm o p opidium iodide de ec ion. Real- ime RT-PCR Quan i a i e eal- ime RT-PCR was used o analyze he exp ession o he MHR5, FLA6, RHD6 and RSL4 genes. To al RNA was ex ac ed om A abidopsis lea es using he Qiagen RNeasy Plan Mini Ki . RNA was e e se ansc ibed using an oligo (dT) p ime and In i ogen Supe -sc ip Fi s -S and Syn hesis Sys em o RT-PCR ollowing he manu ac u e ’s ins uc ions. Gene-speci ic p ime s o each gene we e designed using he In i ogen Vec o NTI Ad ance 10 so wa e. The p ime sequences we e as ollows: QFMRH5, 5´- GCTGCTTGCTGCTCAAATCC-3´ and QRMRH5, 5´-AATCCAGAGAATCCACCACG-3´ o he MRH5 gene; QFFLA6, 5´-CAAATCCAGCTCATGCTCTACC-3´ and QRFLA6, 5´-TCTTGTCCCGTAGCCTGAGT- Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 17 3´ o FLA6 gene; qRHD6-Fw, 5´- CCGGCTCAAGGAGGAAAA-3´ and qRHD6-R, 5´- CGAATTCCTGTCTCGTTGTGA-3´ o RHD6 gene; qRSL4-Fw, 5´- CAGATTAAGTTGTTGAGCTCGG-3´ and qRSL4-R, 5´- GAGACAAAAGGTTGTGATGGAA-3´ o RSL4 gene; qUbq10-F, 5´- GGCCTTGTATAATCCCTGATGAATAAG-3´ and qUbq10-R, 5´- AAAGAGATAACAGGAACGGAAACATAGT-3´ o he cons i u i e UBQ10 gene. Real- ime PCR was pe o med using he Bio-Rad IQ SYBR G een Supe mix. Signals we e de ec ed wi h a Bio-Rad iCYCLER acco ding o he manu ac u e ’s ins uc ions. The cycling p o ile consis ed o 95°C o 10 min ollowed by 45 cycles o 95°C o 15 s and 60°C o 1 min. A mel ing cu e om 60°C o 90°C was pe o med ollowing PCR cycling. The exp ession le els o genes o in e es we e no malized o ha o he cons i u i e UBQ10 gene by sub ac ing he cycle h eshold (CT) alue o UBQ10 om ha o he gene o in e es (∆CT) and calcula ed as 2 - ∆CT . The esul s shown a e he means ± SD o a leas h ee independen RNA samples. De e mina ion o ATP The adenine iphospha e in he oo s was ex ac ed ollowing he boiling wa e me hod (Yang e al. 2002). B ie ly, 50 mg o oo issues we e mixed wi h 100 µl o ice-cold dis illed H 2 O, which was immedia ely hea ed in a boiling wa e ba h o 10 min (Li e al. 2017). The boiled lysa es we e cen i uged a 15,000 g o 5 min a 4 ºC and he supe na an s we e collec ed o ATP measu emen using an ATP De e mina ion ki (A22066, The moFishe Scien i ic®), ollowing he manu ac u e ’s p o ocol in a The mo Scien i ic Va ioskan® Flash. Cyanide de e mina ion by high-pe o mance liquid ch oma og aphy (HPLC) A o al o 100 mg o oo issue was homogenized in liquid ni ogen using a mo a and pes le and esuspended in cold bo a e-phospha e ex ac ion bu e (2 mL g -1 esh weigh ) con aining 27 mM sodium bo a e and 47 mM po assium phospha e, pH 8.0. The homogena es we e cen i uged a 15,000 g o 15 min a 4°C. Ex ac ed cyanide was subsequen ly quan i ied by e e se-phase HPLC a e de i a iza ion wi h 2,3- naph halenedialdehyde o o m a 1-cyano-2-alkyl-benz[ ]isoindole de i a i e using p e iously desc ibed me hods (Ga cia e al. 2010; Lin e al. 2005). De e mina ion o NADPH oxidase ac i i y Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 18 NADPH ac i i y in oo ex ac s was measu ed using a me hod adap ed om a p e ious epo (Kaundal e al. 2012). F ozen oo s (50 mg) we e homogenized in 1 ml o 50 mM HEPES bu e (pH 7.2) con aining 0.25 M suc ose, 3 mM EDTA, 1 mM di hio ei ol (DTT), 3.6 mM L-cys eine, 0.1 mM MgCl 2 and 0.6% poly inylpy olidone (PVP) wi h he addi ion o Comple e P o ease Inhibi o Cock ail Table s (Sigma). The homogena e issue was il e ed h ough wo laye s o Mi aclo h and cen i uged a 10,000 x g o 45 min a 4ºC. The supe na an was cen i uged a 203,000 g o 60 min a 4ºC. The pelle was esuspended in 150 µl ice-cold 10 mM T is-HCl (pH 7.4) and used o he enzyme assay. NADPH oxidase ac i i y was assayed colo ime ically wi h XTT (3′-[1-[(phenylamino)-ca bony]-3,4- e azolium]-bis(4-me hoxy-6-ni o)benzene-sul onic acid) sodium sal as a subs a e. The eac ion mix u e con ained 50 mM T is-HCl bu e (pH 7.5), 0.5 mM XTT, 0.1 mM NADPH and 5 µg p o ein ex ac . The linea inc ease in abso p ion a 492 nm due o he o ma ion o a yellow o mazan was ollowed o 120 min (ex inc ion coe icien o 21.6 mM -1 cm -1 ). Funding This wo k was suppo ed in pa by he Eu opean Regional De elopmen Fund h ough Minis e io de Economía y Compe i i idad [g an MOLCYS, no. BIO2013-44648-P] and Agencia Es a al de In es igación [g an no. BIO2016-76633-P] L.A.-A. hanks he Minis e io de Economía y Compe i i idad o ellowship suppo h ough he p og am o Fo mación de Pe sonal In es igado . Acknowledgmen s We hank D . Alicia O ea o con ocal mic oscopy se ice and Inmaculada Mo eno o echnical assis ance. Re e ences Al a ez, C., Ga cia, I., Rome o, L.C. and Go o , C. 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Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 22 Tables Plan line Adenosine iphospha e (nM) Wild ype 400.9 ± 118.6 cas-c1 427.6 ± 118.2 Table 1. Adenosine iphospha e le els in oo ex ac s. The ATP le el was de e mined in c ude ex ac s p epa ed om he oo s o wild ype and cas-c1 mu an plan s g own in solid MS medium supplemen ed wi h suc ose o 14 d. Values a e means ± SD om h ee independen expe imen s. Plan line NADPH oxidase ac i i y (mU/µg) wild ype 23.23 ± 2.8 cas-c1 20.64 ± 0.6 wild ype + KCN 0.1mM 21.09 ± 0.5 Table 2. NADPH oxidase ac i i y le el in oo ex ac s. The ac i i y was de e mined in c ude ex ac s wi h o wi hou 0.1 mM KCN ea men , p epa ed om he oo s o wild ype and mu an s g own in solid MS medium supplemen ed wi h suc ose o 14 d. Values a e means ± SD om i e independen expe imen s. Legends o igu es Figu e 1. CAS-C1 exp ession and localiza ion s udy. Rep esen a i e GFP images o A,D,G, wild ype, B,E,H, scn1-1 mu an and C,F,I, hd2GK mu an ans o med wi h P oC1:CASC1-GFP. Plan s we e g own in MS medium supplemen ed wi h suc ose o 2 days. Images a e maximum p ojec ion o 20 op ical sec ions. Figu e 2. Roo hai pheno ypes o oo hai o ma ion mu an s. (A, E) Wild ype, (B, F) cas-c1, (C) scn1-1, (D) scn1-1 cas-c1, (G) hd2-1, (H) hd2-1 cas-c1 . Seedlings we e g own 6-d on MS (A-D) o 7-d on MS pH4 (E-H) medium supplemen ed wi h suc ose in e ical pla es. Rep esen a i e images a e shown. Ba s = 0.5 mm. Figu e 3. Hyd oxocobalamin e ec on oo hai o ma ion. (A-D) Roo hai pheno ype o wild ype, cas-c1, scn1-1 and scn1-1 cas-c1 3-d-old plan s, espec i ely, g own on MS suc ose e ical pla es in he absence o hyd oxocobalamin. (E-H) Roo hai pheno ype o wild ype, cas-c1, scn1-1 and scn1-1cas-c1 3-d-old plan s, espec i ely, g own on MS suc ose e ical pla es in he p esence o 5 mM hyd oxocobalamin. (I-L) Roo hai pheno ype o wild ype, cas-c1, hd2-1 and hd2-1 cas-c1 7-d-old plan s, espec i ely, g own on MS pH4 Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 23 suc ose e ical pla es in he absence o 5 mM hyd oxocobalamin. (M-P) Roo hai pheno ype o wild ype, cas- c1, hd2-1 and hd2-1 cas-c1 7-d-old plan s, espec i ely, g own on MS pH4 in he p esence o 5 mM hyd oxocobalamin. All he expe imen s we e epea ed a leas h ee imes, wi h simila esul s ob ained each ime. Ba s = 0.5 mm. Figu e 4. Roo hai o ma ion genes exp ession analysis. Real- ime (RT)–PCR analysis o he exp ession o he RHD6, RSL4, FLA6 and MRH5 genes was pe o med in oo ex ac s om 14-day-old wild- ype, cas-c1, scn1-1 and scn1-1 cas-c1 mu an s g own in MS pH 5,7 (A) o wild- ype, cas-c1, hd2-1 and hd2-1 cas-c1 mu an s g own in MS pH 4 (B). The ansc ip le els we e no malized o he cons i u i e UBQ10 gene. Da a shown a e means ± SD o h ee independen expe imen s. Di e en le e s abo e he ba indica e signi ican di e en mean (ANOVA es , P<0.01). Figu e 5. De ec ion o supe oxide in A abidopsis oo s. (A) Wild- ype, (D) cas-c1, (B) scn1-1, (E) scn1-1 cas- c1, (C) hd2-1 and (F) hd2-1 cas-c1 seedlings we e g own o 3 d on MS medium wi h suc ose. The oo s we e s ained wi h NBT as desc ibed in Ma e ials and Me hods. Rep esen a i e images a e shown. All he expe imen s we e epea ed a leas h ee imes, wi h simila esul s ob ained each ime. Ba s = 0.1 mm. Figu e 6. Accumula ion o H 2 O 2 in wild ype and oo hai mu an s. H 2 O 2 was de ec ed by H 2 DCFDA s aining in 14-d-old oo s o (A) wild- ype, (D) cas-c1, (B) scn1-1, (E) scn1-1 cas-c1, (C) hd2-1 and (D) hd2-1 cas-c1 plan s cul u ed on MS medium supplemen ed wi h 1% suc ose. All he expe imen s we e epea ed a leas h ee imes, wi h simila esul s ob ained each ime. Ba s = 300 µm. Figu e 7. In ol emen o CAS-C1 and cyanide in he oo hai g ow h. In an elonga ing oo hai , SCN1 inhibi s he ROPs p o eins, which a e essen ial o he NADPH oxidase RHD2 ip-localized ac ion. RHD2 p oduces supe oxide anion (s ained wi h NBT) ha is ans o med o oxygen pe oxide ( isualized by H 2 DCFDA s aining) by supe oxide dismu ase ac ion. Bo h ROS a e impo an o cell wall g ow h by b eaking and ebuilding. Cyanide concen a ion, con olled by CAS-C1 and elimina ed by COB, would ac in a s ep be ween he SCN1 ac ion and he ROS p oduc ion by RHD2, es ablishing posi i e (a ows) o nega i e (blun lines) ela ionships o hi he o unknown p o ein(s) o ac o (s). Full lines indica e al eady es ablished ela ionships, while dashed lines indica e he p oposed sequence o ac ion o CAS-C1 and cyanide in he oo hai elonga ion p ocess. Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 24 Supplemen a y Fig. S1. Schema ic ep esen a ion o he CAS-C1 locus and lanking genes on ch omosome 3. A, Diag am o he in e genic sequence be ween CAS-C1 and he adjacen genes on ch omosome 3 o he A abidopsis genome (www.a abidopsis.o g). Yellow a ows indica e he di ec ion o ansc ip ion o he di e en genes. Da k a ows indica e he designed oligonucleo ide posi ions o P oC1:CASC1 sequence isola ion. B, The nucleo ide sequence o P oC1:CASC1, whe e P oC1 is he CAS-C1 p omo e , and CASC1 is he CAS-C1 ORF. Colo ed le e s indica e in on (pu ple), UTR ( ed) and exon (yellow) sequence. Blue shadowed le e s show he ansla ional s a and s op codons. PIP-1 codes o a plasma memb ane in insic p o ein and SYP-73 codes o a syn axin o plan s. Supplemen a y Fig. S2. Mi ochond ial localiza ion o CASC1-GFP (A) Maximum p ojec ion o 20 op ical sec ions o 4-d-old oo issues om wild ype plan s ans o med wi h P oC1:CASC1-GFP showing GFP luo escence signal. (B) Fluo escence signal o he same issues dyed wi h Mi oT acke Deep Red 633 o 15 min, (C) O e lapping o (A) and (B) images showing co-localiza ion o GFP luo escence and s ained mi ochond ia. Ba s = 40 µm. Supplemen a y Fig. S3. Roo pheno ype o he cas-c1 and complemen ed mu an line. (A) Rep esen a i e b igh - ield image o 4-day-old cas-c1 mu an plan s is shown. (B-C) Rep esen a i e b igh - ield and GFP luo escence images, espec i ely, o 4-day-old cas-c1 mu an plan s ans o med wi h P oC1:CASC1-GFP a e shown. Images a e maximum p ojec ion o 20 op ical sec ions. Ba s = 50 µm Supplemen a y Fig. S4. Molecula cha ac e iza ion o he scn1-1 cas-c1 mu an . To iden i y indi iduals homozygous o SCN1 and CAS-C1 gene mu a ions, genomic DNA was ex ac ed om lea es o wild- ype and eigh scn1-1 cas-c1 mu an s g own in MS medium supplemen ed wi h suc ose (1%) and kanamycin (30 µg/mL) and ansplan ed o soil la e . This DNA was subjec ed o he ollowing: A, PCR geno yping using he p ime pai s scn1-1 FW/scn1-1 REV and C1-F2/C1-R2, wi h UBQ10 ampli ica ion using UBQF1-UBQR1 as a posi i e con ol; B, sequencing geno yping using scn1-1 FW/scn1-1 REV p ime s. scn1-1 FW: 5´-TCAAAGAACATCTCGAGAAGGA-3´ scn1-1 REV: 5´-CACACAAACACACCTCCAATGT-3´ C1-F2: 5´-TGATGGGAATTGGCAGTGGAGGCAC-3´ C1-R2: 5´-AATTGCTTGCCACGGTGTTAGCTCCC-3´ Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 25 UBQF1: 5’- GATCTTTGCCGGAAAACAATTGGAGGATGGT-3’ UBQR1: 5’- CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3’ Supplemen a y Fig. S5. Molecula cha ac e iza ion o he hd2-1 cas-c1 mu an . To iden i y indi iduals who we e homozygous o RHD2 and CAS-C1 gene mu a ions, genomic DNA was ex ac ed om lea es o wild- ype, hd2-1 mu an (as posi i e con ol) and i e hd2-1 cas-c1 mu an s g own in MS medium supplemen ed wi h suc ose (1%) and kanamycin (30 µg/mL) and ansplan ed o soil la e . This DNA was subjec ed o he ollowing: A, PCR geno yping using he p ime pai s hd2-1 FW/ hd2-1 REV and C1-F2/C1-R2, wi h UBQ10 ampli ica ion using UBQF1-UBQR1 was used as a posi i e con ol; B, sequencing geno yping using hd2-1 FW/ hd2-1 REV p ime s. hd2-1 FW: 5´-TGAATGGTATGAACCAAACCGC-3´ hd2-1 REV: 5´-GGCTTACACACCTGAAACAACA-3´ Supplemen a y Fig. S6. 1-Aminocyclop opane-1-ca boxylic acid (ACC) e ec on oo hai o ma ion. (A, C, E, G, I, K) Roo hai pheno ype o wild ype, cas-c1, scn1-1, scn1-1cas-c1, hd2-1 and hd2-1 cas-c1 6-d-old plan s, espec i ely, g own on MS suc ose e ical pla es in he absence o ACC. (B, D, F, H, J, L) Roo hai pheno ype o wild ype, cas-c1, scn1-1, scn1-1cas-c1, hd2-1 and hd2-1 cas-c1 6-d-old plan s, espec i ely, g own on MS suc ose e ical pla es in he p esence o 50 µM ACC. Ba s = 0.15 cm. Supplemen a y Fig. S7. Cyanide de e mina ion in oo issues. Fou een-days old wild ype, cas-c1, scn1-1, scn1-1 cas-c1, hd2-1 and hd2-1 cas-c1 mu an plan s we e g own on pla es con aining MS suc ose medium and hen collec ed o cyanide con en de e mina ion. Values a e he means ± SD om i e independen expe imen s. Inc eases o he media o he cyanide con en in e e y couple o mu an s a e indica ed a he op o he espec i e column pai . As e isks indica e signi ican di e ences (ANOVA es , P<0,01) be ween wild ype and cas-c1 and be ween pai s o single and double mu an s. Supplemen a y Video S1. Roo hai g ow h. P oC1:CASC1-GFP plan s we e g own o 2 d and luo escence mic oscopy images we e aken e e y 2 min o e 2 h o analyze oo hai o ma ion in i o. Ba = 25 µm. Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018 SCN1 CAS-C1 ROPs RHD2 O2.- H2O2 [CN-] COB NBT s aining H2DCFDA s aining Fig. 7. In ol emen o CAS-C1 and cyanide in he oo hai g ow h. In an elonga ing oo hai , SCN1 inhibi s he ROPs p o eins, which a e essen ial o he NADPH oxidase RHD2 ip-localized ac ion. RHD2 p oduces supe oxide anion (s ained wi h NBT) ha is ans o med o oxygen pe oxide ( isualized by H2DCFDA s aining) by supe oxide dismu ase ac ion. Bo h ROS a e impo an o cell wall g ow h by b eaking and ebuilding. Cyanide concen a ion, con olled by CAS-C1 and elimina ed by COB, would ac in a s ep be ween he SCN1 ac ion and he ROS p oduc ion by RHD2, es ablishing posi i e (a ows) o nega i e (blun lines) ela ionships o hi he o unknown p o ein(s) o ac o (s). Full lines indica e al eady es ablished ela ionships, while dashed lines indica e he p oposed sequence o ac ion o CAS-C1 and cyanide in he oo hai elonga ion p ocess. Page 32 o 32Plan & Cell Physiology Downloaded om h ps://academic.oup.com/pcp/ad ance-a icle-abs ac /doi/10.1093/pcp/pcy047/4911869 by Cen o de In o mación y Documen ación Cien í ica, [email p o ec ed] on 01 Ma ch 2018