Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines

RESEARCH ARTICLE Phenolic Compounds and Exp ession o 4CL Genes in Sil e Bi ch Clones and P 4CL1a Lines Su i Su ela 1 *, Te hi Hahl 1¤ , Heidi Tiimonen 2 , Tuija A onen 3 , Tiina Ylioja 4 , Tapio Laakso 4 , Pekka Sa anpa¨a¨ 4 , Vincen Chiang 5 , Rii a Julkunen-Tii o 6 , Hely Ha¨ggman 1 1. Depa men o Biology, Uni e si y o Oulu, Oulu, Finland, 2. The Finnish Bo de Gua d, Bo de and Coas Gua d Academy, Ima a, Finland, 3. Finnish Fo es Resea ch Ins i u e, Eas e n Finland Regional Uni (Punkaha ju Uni ), Punkaha ju, Finland, 4. Finnish Fo es Resea ch Ins i u e, Sou he n Finland Regional Uni (Van aa Uni ), Van aa, Finland, 5. Fo es Bio echnology G oup, Depa men o Fo es y and En i onmen al Resou ces, No h Ca olina S a e Uni e si y, Raleigh, No h Ca olina, Uni ed S a es o Ame ica, 6. Depa men o Biology, Uni e si y o Eas e n Finland, Joensuu, Finland *[email p o ec ed] ¤ Cu en add ess: Ins i u e o E olu iona y Biology and En i onmen al S udies, Uni e si y o Zu¨ ich, Zu¨ ich, Swi ze land Abs ac A small mul igene amily encodes 4-couma a e:CoA ligases (4CLs) ca alyzing he CoA liga ion o hyd oxycinnamic acids, a b anch poin s ep di ec ing me aboli es o a la onoid o monolignol pa hway. In he p esen s udy, we examined he e ec o an isense Populus emuloides 4CL (P 4CL1) o he lignin and soluble phenolic compound composi ion o sil e bi ch (Be ula pendula) P 4CL1a lines in compa ison wi h non- ansgenic sil e bi ch clones. The endogenous exp ession o sil e bi ch 4CL genes was eco ded in he s ems and lea es and also in lea es ha we e mechanically inju ed. In one o he ansgenic P 4CL1a lines, he a io o sy ingyl (S) and guaiacyl (G) lignin uni s was inc eased. Mo eo e , he ansc ip le els o pu a i e sil e bi ch 4CL gene (Bp4CL1) we e educed and con en s o cinnamic acid de i a i es al e ed. In he o he wo P 4CL1a lines changes we e de ec ed in he le el o indi idual phenolic compounds. Howe e , conside able a ia ion was ound in he ansc ip le els o sil e bi ch 4CLs as well as in he concen a ion o phenolic compounds among he ansgenic lines and non- ansgenic clones. Wounding induced he exp ession o Bp4CL1 and Bp4CL2in lea es in all clones and ansgenic lines, whe eas he ansc ip le els o Bp4CL3 and Bp4CL4 emained unchanged. Mo eo e , mino changes we e de ec ed in he concen a ions o phenolic compounds caused by wounding. As an o e all end he wounding dec eased he la onoid con en in sil e bi ches and inc eased he con en o soluble condensed annins. The esul s indica e ha by educing he OPEN ACCESS Ci a ion: Su ela S, Hahl T, Tiimonen H, A onen T, Ylioja T, e al. (2014) Phenolic Compounds and Exp ession o 4CL Genes in Sil e Bi ch Clones and P 4CL1a Lines. PLoS ONE 9(12): e114434. doi:10.1371/jou nal.pone.0114434 Edi o : Lou des Go´mez-Go´mez, UCLM, Spain Recei ed: Sep embe 3, 2014 Accep ed: No embe 10, 2014 Published: Decembe 11, 2014 Copy igh : ß2014 Su ela e al. This is an open- access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep o- duc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y: The au ho s con i m ha all da a unde lying he indings a e ully a ailable wi hou es ic ion. All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: The esea ch was unded by he Academy o Finland (P ojec 104532 o HH), Jenny and An i Wihu i Founda ion, and Niemi Founda ion ( o SS) and by he Biological Socie y o Finland Vanamo and Na u al His o y Socie y o Oulu ( o TH). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 1/29 Bp4CL1 ansc ip le els lignin composi ion could be modi ied. Howe e , he al e a ions ound among he P 4CL1a lines and he non- ansgenic clones we e wi hin he na u al a ia ion o sil e bi ches, as shown in he p esen s udy by he clonal di e ences in he ansc ip s le els o 4CL genes, soluble phenolic compounds and condensed annins. In oduc ion Phenolic compounds o m a di e se g oup o seconda y me aboli es exhibi ing impo an oles in plan de elopmen and en i onmen al adap a ion [1–3]. A benzene ing a ached o a leas one hyd oxyl g oup is a common cha ac e is ic o phenolic compounds, mos ly de i ed om cinnamic acid, gene a ed a he i s s ep o he gene al phenylp opanoid pa hway. Ca bon low h ough he gene al phenylp opanoid ou e is ex ensi e as he hyd oxycinnamic acids a e u ilized in he monolignol biosyn he ic pa hway, gi ing ise o he second mos abundan o ganic compound on ea h, lignin. The accumula ion o lignin in seconda y cell walls o ascula plan s p o ides mechanical s eng h, p o ec ion agains pa hogens and he bi o es, and enables he essen ial anspo a ion o wa e and solu es. Fla onoids, hyd oxycinnamic acids, phenolic glycosides, and condensed annins (CTs) ep esen o he in ensi ely s udied phenolics ha ing unc ions in allelopa hy, pollen e ili y, auxin anspo , pollina o a ac ion and in de ence agains pa hogens, pes s, UV-B adia ion, and oxida i e damage [1–3]. Re lec ing he di e gen oles o phenolic compounds, he gene al phenylp o- panoid ou e can be ac i a ed by abio ic and bio ic s ess ac o s, such as cold, ozone, CO 2 , ligh , pa hogens, and he bi o es [4]. In addi ion, he phenylp opa- noid ou e is igge ed by mechanical wounding as upon damage he wound si e needs o be sealed in p e en ion o dehyd a ion and possible pa hogen in ec ions. A he wound si e he cell walls a e i s s eng hened by c osslinking p o eins. Phenylp opanoid de i a i es a e equi ed in he subsequen s ep, ligni ica ion and sube iza ion o he cell walls [5]. Fu he mo e, mechanical wounding can induce p oduc ion o phenolic compounds which a e gene ally conside ed o in e ac in he de ence esponses. Howe e , simila o induced de ences caused by he bi o y esponse, also he accumula ion o phenolic seconda y me aboli es is species dependen [6,7]. The gene al phenylp opanoid ou e is ini ia ed by he deamina ion o phenylalanine by phenylalanine ammonia-lyase (PAL; EC 4.3.1.5). In he ollowing s ep cinnamic acid can be con e ed o p-couma ic acid by cinnamic acid 4-hyd oxylase (C4H; EC 1.14.13.11). Finally, as he las s ep o he gene al phenylp opanoid ou e, 4-couma a e:CoA ligase (4CL; 6.2.1.12) ca alyzes he o ma ion o p-couma oyl-CoA. In addi ion o p-couma ic acid, he 4CL iso o ms a e able o u ilize a a ie y o hyd oxycinnamic acid de i a i es as subs a es, and posi i e connec ion has been ound be ween he subs a e di e si y o 4CLs and 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 2/29 he ex en o gene amily expansion [8]. Howe e , he 4CL gene amilies a e ela i e small, o ins ance in A abidopsis he numbe o 4CLs is h ee [9–11]. The angiospe m 4CLs a e ca ego ized o wo classes: class I and class II [9,12,13]. Mos o cha ac e ized 4CLs belong o class I, he membe s o which a e gene ally conside ed o unc ion in he lignin biosyn hesis [9,13–20]. In addi ion, he 4CL gene amilies o en include 4CLs ha ing dis inc subs a es such as sinapa e [12,21] and 4CL-like genes, gene ally conside ed nei he o be connec ed wi h ligni ica ion no la onoid biosyn hesis [9,10,22,23]. In he phenylp opanoid ou e, 4CL is conside ed as a b anching poin a which he me abolic luxes a e di ec ed ei he o la onoid o monolignol biosyn he ic pa hway. The monolignol biosyn he ic pa hway p oduces lignin monome s, coni e yl -, sinapyl - and p-couma yl alcohol, in cy oplasm and subsequen polyme isa ion o monolignols wi h polysaccha ides occu s in he plan cell wall. In ha dwoods lignin is composed o guaiacyl (G), sy ingyl (S) and p- hyd oxyphenyl (H) uni s ha ing di e en me hyla ion deg ees which esul s in a ying linkage ypes in he polyme . Hence, he s uc u al complexi y and chemical solubili y o lignin depend on i s monolignol composi ion. The composi ion and con en o lignin can be al e ed by modi ying he exp ession o monolignol biosyn he ic pa hway genes [11,24]. By educing he exp ession o pa icula 4CL genes, he lignin con en has been dec eased in A abidopsis [11,16], Populus emuloides [25,26], P. omen osa [27,28], P. emula 6alba [29], P. ichoca pa [30], Nico iana abacum [31], ice (O yza sa i a)[20], and Pinus adia a [32]. In addi ion o he lignin con en , also he lignin composi ion [16,28,29,32,33] and concen a ion o phenolics [11,20,25,28,29,32,34] ha e been shown o al e as a esul o educed 4CL exp ession. Sil e bi ch (Be ula pendula Ro h) has been unde ac i e esea ch in Finland due o i s economic impo ance and cen al ole in he bo eal o es ecosys ems [35]. Besides he con en ional b eeding me hods, he mode n molecula me hods ha e been shown o be applicable o sil e bi ch [35,36]. Fu he mo e, he possible en i onmen al isks associa ed o gene ically modi ied (GM) ees, ha e been s udied wi h sil e bi ch lines ha ing in oduced ai s a ge ed o modi y he lignin cha ac e is ics [37–40] and disease esis ance [41–46]. In gene al, modes pleio opic e ec s ha e been de ec ed in he s udies wi h GM sil e bi ch lines. In he p esen wo k phenolic compounds and he exp ession o endogenous 4CLs a e s udied in ou sil e bi ch clones. Sil e bi ch lines exp essing an isense quaking aspen (Populus emuloides L.) P 4CL1 gene, we e gene a ed and cha ac e ized and he e ec o mechanical wounding on soluble phenolic compounds and 4CL gene exp ession was de e mined. Ma e ials and Me hods Gene a ion o P 4CL1a sil e bi ch lines Th ee ansgenic lines ep esen ing he sil e bi ch (Be ula pendula Ro h) geno ypes A, E5382, and E5396 we e gene a ed by ans o ma ion wi h P 4CL1 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 3/29 (AF041049), he gene de i ed om quaking aspen (P. emuloides). The clone A ep esen s he p ogeny om he c osses be ween sil e bi ch lines o sou he n Finnish o igin, E1970 (Kangasala) and E1980 (Nummi-Pusula). E5382 and E5396 we e cul i a ed in he clonal a chi e in Punkaha ju (61˚499N; 29˚189E). The P 4CL1 exp essing sil e bi ch lines we e gene a ed by he means o biolis ic ans o ma ion wi h he PDS-1000/He de ise (Bio-Rad Labo a o ies Inc., He cules CA) using in i o s em pieces as explan s as desc ibed in Valjakka e al. [36]. The gene cons uc pRT9/35S-P COMT [47] was used as a backbone o he cons uc ion o pRT99/35S-P 4CL1-a plasmid ec o . The pRT9/35S-P COMT con ained he neomycin phospho ans e ase II (np II) gene unde he con ol o he CaMV 35S p omo e and he P COMT agmen ollowed by he NOS e mina o d i en also by he CaMV 35S p omo e . The pRT99/35S-P 4CL1-a (S1 Figu e in S1 File) was gene a ed by subs i u ing he P COMT sequence wi h he P 4CL1 using he XbaI and BamHI es ic ion si es. The 1.9 kb long agmen o P 4CL1 was liga ed o he p omo e and NOS e mina o in an isense o ien a ion and he accu acy o cons uc was con i med by sequencing. The selec ion o ans o med sil e bi ch ma e ial was based on he usage o 100–200 mg/L kanamycin (kan) in he Woody Plan Medium (WPM, [48]) a e a week’s cul i a ion on an ibio ic ee WPM [47]. The egene a ion o indi idual sil e bi ch lines, as well as oo ing, was accomplished on he WPM as desc ibed in A onen e al. [47]. The in eg a ion o he gene cons uc and he exp ession o ansgenes we e con i med using Sou he n and No he n blo analysis. The DNA ex ac ion was pe o med as desc ibed in Valjakka e al. [36], and Sou he n blo analysis as desc ibed in A onen e al. [47]. The es ic ion enzymes XbaI and BamHI we e used in he diges ion o 15 mg genomic DNA. The digoxigenin-11-dUTP labelled p obes speci ic o np II and P 4CL1 we e gene a ed wi h PCR eac ion desc ibed in A onen and Ha ¨ggman [49] wi h p ime s p esen ed in S1 Table in S2 File. The p ocedu e o o al RNA isola ion and o he No he n blo can be ound in A onen e al. [47]. Cha ac e iza ion o he P 4CL1a sil e bi ch lines The i s expe imen al se -up included he ansgenic lines A1, A2, A5 and E5382/ 3 de i ed om clones A and E5382, espec i ely. The second expe imen al se -up consis ed o clone E5396 and he ansgenic line E5396/4. Bo h expe imen s we e conduc ed a he g eenhouse o Finnish Fo es Resea ch Ins i u e Punkaha ju Uni unde s anda d g eenhouse condi ions and na u al ligh condi ions. In he i s expe imen al se -up, he po ed plan s o A, A1, A2, A5, E5382 and E5382/3 we e placed in o ou eplica es in June whe e 80 plan s we e placed in andomly assigned design. To a oid po en ial edge e ec s hey we e su ounded by addi ional plan s no used as expe imen al ma e ial. The numbe o indi idual plan s o clones E5382 and A and he ansgenic lines A1, A2, and A5 was equal, ha is, 13 indi iduals in he h ee eplica es and 12 indi iduals in he ou h. The numbe o plan s o ansgenic line E5382/3 was 11 in he h ee eplica es and 10 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 4/29 in he ou h. Fu he mo e, he h ee eplica es included h ee, and he ou h eplica e 13 addi ional bi ches su ounding he s udied plan s. The mo phology, g ow h cha ac e is ics and phenology o he sil e bi ches we e eco ded o wo g owing seasons a e which nine indi idual sil e bi ches pe line/clone we e selec ed o be moni o ed and g own o addi ional hi d g owing season. The s em and lea samples o lignin and No he n blo analyses we e collec ed du ing second g owing season. The lea es we e collec ed below he la es shoo and ma u e lea es in sho shoo s a he base o la e al shoo s we e used. Pa o he collec ed lea es was used in expe imen s wi h he bi ou s la ae. The expe imen wi h clone E5396 and ansgenic line E5396/4 consis ed o wo eplica es. Each expe imen was composed o 30 po ed indi idual plan s ep esen ing clone E5396 and line E5396/4 and hey we e o ganized in o pa allel, andomly assigned design. The wo eplica e expe imen s con ained al oge he 120 plan s and su ounded by addi ional bi ches no used as expe imen al ma e ial. The expe imen s a ed in Augus . A he end o he second g owing season, inal g ow h pa ame e s we e eco ded, and samples aken o analyses. The Klason lignin measu emen s we e conduc ed o 2-yea -old s em samples ep esen ing all bi ch clones and lines as desc ibed in A onen e al. [47]. Besides, he de e mina ion o S and G moie ies was conduc ed wi h 2-yea -old s em samples, howe e , wi hou samples om he clone E5396 and he ansgenic line E5396/4. The p ocedu e and he ch oma og aphic condi ions we e ca ied ou as desc ibed by Tiimonen e al. [37]. Tes wi h lepidop e an la ae The ood quali y o lea es we e examined by o e ing he lea es o la ae o h ee geome ids Ae halu a punc ula a Denis & Schi ., Cleo a cinc a ia Denis & Schi ., Epi i a au umna a Bo k., and noc uid O hosia go hica L. in a no-choice es . The adul s o A.punc ula a,C.cinc a ia, and O.go hica we e cap u ed in sou h-eas e n Finland and la ae o E.au umna a we e collec ed in no he n Finland. La ae we e ea ed on die consis ing o B.pendula. O he ou plan s pe eplica e o clones A and E5382 and lines A1, A2, A5, and E5382/3 we e andomly selec ed and lea es collec ed o he RGR expe imen . The ull-g own sho shoo lea es we e collec ed om he la e al shoo s. Each la a was weigh ed and posi ioned on a lea which was on a mois il e pape on a Pe i dish o 9 cm o diame e . The expe imen con inued o 24 h which a e he la ae we e weigh ed again. Fo A. punc ula a,C.cinc a ia and E.au umna a he expe imen was epea ed using he same plan indi iduals (se ies I and II). The RGR alues ([ln(end weigh ) - ln(ini ial weigh )]/d) o la ae we e excluded when no weigh gain was eco ded o when la ae had died/moul ed o appea ed o he wise unwell. In addi ion, when la ae had consumed whole o almos whole lea o i he lea had begun yellowing, alues we e no included in he analysis. 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 5/29 Wounding expe imen The plan ma e ial consis ed o ou non- ansgenic sil e bi ch clones (A, R, E5382, E5396) and ou ansgenic lines (A1, A5, E5382/3, E5396/4). The non- ansgenic clone R ep esen s a p ogeny om a c oss be ween egis e ed sil e bi ches V5411 and V5402. The sil e bi ch ma e ial was mul iplied a 21–22˚C wi h ligh /da k pho ope iod o 16 h/8 h (110–130 mmol m 22 s 21 ) on WPM supplemen ed wi h 2.2 mM 6-benzyladenine (BA) and 2.8 mM indole-3-ace ic acid (IAA), and accompanied wi h 200 mg/L kan o he ansgenic sil e bi ch lines. The oo ing o plan le s was conduc ed on WPM wi hou plan g ow h egula o s o an ibio ics. The 5-week acclima iza ion pe iod o plan s ook place a he Bo anical Ga dens o he Uni e si y o Oulu on a mix u e o Kekkila ¨Seedling Soil (Kekkila ¨Oy, Finland) and sand. The plan s we e ansplan ed in Ma ch and placed in a andomly assigned expe imen al design in he beginning o Ap il. The numbe o indi idual plan s in he expe imen al design a ied pe clone/line be ween 70 and 120. The wounding o plan s was conduc ed du ing Ap il and May by mechanically c ushing app oxima ely 20% o he lea ma gins o wo adjacen lea es in each plan using plie s. The plan s o simila heigh s we e selec ed o he con ol and wounding ea men s. The lea plas och on index (LPI) o he wo wounded lea es anged om LPI 2 o LPI 4 when he i s ully expanded lea was conside ed as LPI 0. When coun ed om he plan apex he posi ion o LPI 2–4 lea es anged be ween he se en h and ele en h lea , depending on he plan indi idual. The samples we e aken immedia ely, and 1, 3, 12, 24, 72, and 168 hou s a e he wounding o he de e mina ion o ansc ip le els. Fo he analysis o phenolic compounds, he samples we e collec ed 21 d a e he wounding. A e e y sampling in ac con ol samples we e also collec ed. The s em heigh s and esh weigh s o all plan s and samples we e eco ded a sample collec ion. The a e age s em heigh (¡SD) o clones/lines we e 15.5¡3.1, 16.8¡3.6, 16.2¡3.5, 17.9¡4, 22.8¡4, 14.5¡5.1, 15.1¡3, and 17.8¡3.5 cm o A, A1, A5, E5382, E5382/3, E5396, E5396/4, and R, espec i ely. Isola ion o ull-leng h 4CL and 4CL-like genes The sequencing o sil e bi ch 4CL genes was based on he EST da a ob ained om Hela iu a and Kauppinen (Uni e si y o Helsinki, Finland). The ESTs we e aligned and sea ched agains he Na ional Cen e o Bio echnology In o ma ion (NCBI) da abase. The p ime s we e designed o expec ed coding sequences (cds) o he ou 4CL genes wi h P ime 3 [50,51] and used in s anda d PCR uns o in addi ional uns wi h he SMART RACE cDNA Ampli ica ion Ki (Clon ech Labo a o ies, Inc., Moun ain View, Cali o nia, US) in acco dance wi h he manu ac u e ’s ins uc ions. The PCR p oduc s ep esen ing he ull cds o pu a i e 4CL genes we e p oduced wi h p ime s p esen ed in S1 Table in S2 File and pu i ied wi h Nucleo Spin Ex ac II Ki (Mache ey-Nagel GmbH & Co. KG, Du ¨ en, Ge many). Subcloning was conduc ed wi h TOPO TA Cloning (In i ogen) and sequencing wi h he BigDye Te mina o 3.1 Cycle Sequencing 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 6/29 Ki (Applied Biosys ems, Fos e Ci y, CA, USA) and he ABI PRISM 377 DNA sequence (Pe kin-Elme , Wellesley, MA, USA). Phylogene ic analysis The phylogene ic analyses we e conduc ed using MEGA6 [52]. The cds o Maniho esculen a (Mes4CL1-4), Glycine max (Gm4CL1-9), Rubus idaeus (Ri4CL1-3), P. emuloides (P 4CL1 and P 4CL2), Be ula pla yphylla (Bpl4CL1), and Be ula lumini e a (Bl4CL1) we e used in phylogene ic analyses. In addi ion, a selec ion o he 4CL and 4CL-like acyl-CoA syn he ase (ACS) genes o A abidopsis, Populus ichoca pa and ice [23] we e included in o he analysis. All used sequences a e p esen ed in S5 Table in S3 File. The 4CL cds we e aligned wi h MUSCLE [53]. The Model Selec ion ea u e was used o e alua e he subs i u ion models o maximum-likelihood (ML) me hod. A phylogene ic ee was econs uc ed using he ML algo i hm [54] and Tamu a 3-pa ame e subs i u ion model [55] wi h he gamma dis ibu ed wi h in a ian si es model. All codons we e included, and pa ial dele ion was used o posi ions con aining missing da a o gaps as ecommended by Hall [56]. The con idence o ML ees was e alua ed wi h boo s ap me hod using 500 eplica es [57] and boo s ap alues anging be ween 70 and 100% we e conside ed eliable [58]. De e mina ion o 4CL ansc ip le els F om lea ma e ial he RNA was ex ac ed using he p o ocol o Jaakola e al. [59], whe eas om s ems he RNA isola ion was conduc ed wi h he GeneJET Plan RNA Pu i ica ion Mini Ki (The mo Scien i ic, Wal ham, Massachuse s, USA) using he p o ocol o RNA pu i ica ion om ligni ied, polyphenol- ich plan issues. Be o e ea ing he RNA wi h DNase I (The mo Scien i ic) RNA was cha ac e ized wi h aga ose gel elec opho esis and ND-1000 UV-Vis Spec opho ome e (NanoD op Technologies, Wilming on, USA). The Re e Aid P emium Re e se T ansc ip ase (The mo Scien i ic) was used in he syn hesis o cDNA, which was dilu ed 1:50 o he eal- ime RT-PCR uns. The eal- ime RT- PCR eac ions o 14 mL consis ed o 1 x Ligh Cycle 480 SYBR G een I Mas e (Roche Applied Science, Penzbe g, Ge many), 0.5 mM o each p ime (S1 and S2 Tables in S2 File) and 3.5 mL o cDNA sample and we e un as duplica es wi h Ligh Cycle 480 (Roche Applied Science). The PCR p og am consis ed o incuba ion a 95˚C o 5 min ollowed by 45 cycles: 10 s a 95˚C, 10 s a 60˚C and 10 s a 72˚C. The mel ing cu e analysis o he Ligh Cycle 480 so wa e and sequencing o he eal- ime RT-PCR p oduc s, using he ABI PRISM 377 DNA sequence (Pe kin-Elme ) and chemis y o he BigDye Te mina o 3.1 (Applied Biosys ems) we e used o con i m he speci ici y o he p ime s. The alpha- ubulin (A ub, AJ279695) and pu a i e p o ein phospha ase 2A egula o y subuni (PP2A, FJ667540) we e used as e e ence genes wi h p ime s desc ibed in Su ela e al. [40]. Se ial dilu ions o pooled cDNA we e used o c ea e p ime speci ic e iciencies o s em and lea samples (S1 and S2 Tables in S2 File). The Abs 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 7/29 Quan /2nd De i a i e Max o All Samples Analysis o Ligh cycle 480 So wa e elease 1.5.0 SP3 was u ilized o gene a e he c ossing poin (Cq) and concen a ion alues (S3 and S4 Tables in S3 File). I Cq could no be de e mined he concen a ion alue was conside as 0. The pe o mance o echnical eplica es was e alua ed and, when necessa y, samples we e e un (see S2 Figu e and S3 Figu e in S1 File o he coe icien a ia ion, CV%, o echnical eplica es). The ela i e exp ession alues we e ob ained using ela i e quan i ica ion wi h ex e nal s anda ds (Roche Applied Science Technical No e No. LC 13/2001) and he ela i e exp ession ep esen s he a ios o a ge and e e ence genes. Fo he de e mina ion o wounding esponse, he ela i e exp ession alues we e di ided by he mean alues o in ac con ol lea es wi hin each clone/line. The A ub and PP2A we e u ilized as e e ence genes as no end was obse ed in he Cq alues due o he wounding ea men (S4 Figu e in S1 File). Analysis o soluble phenolic compounds and CTs The lea and s em samples we e d ied a 60˚C o 48 h, a e which hey we e s o ed a 220˚C. The pooled samples o lea (8 mg) we e cu wi h a co k bo e wi hou he main and la e al eins and, in he case o wounded lea es, also wi hou he damaged lea a ea. O each plan indi idual a pooled s em sample o 15 mg p esen ing only un-wounded plan s was used in he analysis o phenolic compounds. The lea es and s ems we e homogenized wi h P ecellys 24 homogenize (Be in Technologies, Mon igny-le-B e onneux, F ance) wi h 600 mL o me hanol placed in o each P ecellys homogeniza ion ial. The homogeniza ion was conduc ed a 2800 g o 20 s ollowed by 15 min incuba ion on ice. The homogeniza ion s ep was epea ed (2800 g o 20 s) a e which he samples we e cen i uged a 19 000 g o 3 min. Supe na an s we e collec ed and he ex ac ion was epea ed h ee mo e imes using 5 min incuba ions on ice. The combined supe na an s we e d ied in a acuum concen a o (Concen a o 5031, Eppendo , Hambu g, Ge many) a 45˚C o 1 h. The ex ac ion esidues we e d ied o 2 d a oom empe a u e o CT analyses. The dissol ing o samples was conduc ed wi h 600 mL o wa e :me hanol (1:1, / ) and analyzed by HPLC (Agilen 1100 Se ies HPLC Value Sys em, Agilen Technologies, San a Cla a, Cali o nia, US), wi h a diode a ay de ec o (DAD) and Zo bax RRHD SB-C18 column (2.1 mm650 mm, 1.8 mm, Agilen Technologies). The injec ion olume o s em samples was 15 mL and o un-wounded lea samples 20 mL and o wounded 10 mL, which was decided based on he i s HPLC uns, indica ing a high amoun o annin p ecu so s in he wounded lea samples. The iden i ica ion o compounds was based on hei e en ion imes and spec al cha ac e is ics [60]. The comme cial s anda d used in he quan i ica ion o compounds and he conduc ance o CT analyses can be ound in Su ela e al. [40]. 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 8/29 S a is ical analysis The s a is ical examina ion o he da a was ca ied ou using he R 2.11.0 so wa e [61] wi h he g aphical use in e ace, he R Commande [62]. The g ow h and he lignin cha ac e is ics be ween clones and lines we e examined using pai wise compa isons ( he independen -samples es , Welch Two Sample es o Wilcoxon es ). In he case o clone E5396 and line E5396/4 s a is ical es ing was no conduc ed o lignin as he numbe o biological eplica es was wo. The K uskal-Wallis es was used o es o di e ences in RGRs o la ae ed wi h among lea es o sil e bi ch clones and lines. The beginning o au umn lea senescence e.g. he yellowing o lea es be ween ansgenic line (A1, A2 o A5) and non- ansgenic clone A, was s a is ically examined wi h Pea son’s Chi-squa ed es . The indi idual plan s we e ini ially classi ied o A) lea es a e g een; B) some yellowing lea es; C) less han 50% o lea es a e yellowing; D) mo e han 50% o lea es a e yellowing. To ul il he equi emen s o Pea son’s Chi-squa ed es he classes A and B and he classes C and D we e combined when he da a eco ded a he 26 h o Sep . was es ed. When he da a eco ded a he 2 nd o Oc . was es ed he classes A and B and C we e combined. The ela i e exp ession o Bp4CL genes was examined by pai wise compa isons: he independen -samples es , Welch Two Sample es o Wilcoxon es in he case o abno mally dis ibu ed da a. The Bon e oni co ec ion was u ilized o he pai wise es ed ela i e exp ession di e ences o clones. The numbe o biological eplica es was be ween 11–16 in he lea samples and 3–5 in he s em samples wi hin clone/line. The di e ences in phenolic compounds and CTs o lea es we e examined wi h he one-way Ano a and Tukey Con as s o he A, E5382, E5396 and R clones and lines A1, A5, and E5396/4 (n511). Squa e oo and log(x+1) ans o ma ions we e conduc ed o some o he a iables. In addi ion, i he a iable did no mee he assump ions equi ed o Ano a, he es ing was conduc ed i s wi h he K uskal-Wallis ollowed wi h he wo-sample es , Welch Two Sample es o Wilcoxon ank sum es . S a is ical es ing o line E5382/3 wi h lowe numbe o biological eplica es (n58) was conduc ed wi h he wo-sample es , Welch Two Sample es o Wilcoxon ank sum es . The s a is ical analyzes o s em phenolics and CTs (n55) we e he same as used o he lea samples, howe e , in addi ion o line E5382/3, also he clone E5396 was examined wi h he wo-sample es , Welch Two Sample es o Wilcoxon ank sum es because o he low numbe o biological eplica es (n53). The Bon e oni co ec ion was u ilized o he P- alues o pai wise es ed a iables in case o clones. The s a is ical es ing o wounding e ec wi hin clone/line be ween he in ac and wounded lea es collec ed 21 d a e he ea men was conduc ed wi h he wo-sample es , Welch Two Sample es o Wilcoxon ank sum es (n53–8). 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 9/29 concen a ions o cinnamic acids, apigenin de i a i e, salid oside, and soluble CTs (Fig. 3A, S9 Table in S5 File). In gene al, g ea e a ia ion was ound in he phenolic compounds among clones in lea es han in s ems. Clones A and R showed speci ic phenolic p o iles, whe eas he phenolic con en s o clones E5382 and E5396 we e mo e simila (Fig. 3B, S10 Table in S5 File). The cinnamic acid and p-OH-cinnamic acid de i a i es we e bo h signi ican ly (P,0.05) highe in clone R han in o he clones. In addi ion, he concen a ions o kaemp e ol and my ice in 3- hamno- sides and apigenin de i a i es we e signi ican ly (P,0.05) highe in clone R. Kaemp e ol, my ice in and que ce in 3-ace yl-glucosides, as well as que ce in de i a i e, we e de ec ed in clone A bu ound absen o a e y low concen a ions in clones E5382, E5396 and R. In addi ion, he lea es o clone A Fig. 3. The con en s o phenolic compounds and condensed annins in sil e bi ch (Be ula pendula) s ems and lea es. S ems (A) and lea es (B) o clones A, E5382, E5396, and R, and P 4CL1a lines A1, A5 and E5282/3 and np II line E5396/4. Values a e mg/DW g mean ¡SE. Di e en le e s abo e he columns deno e signi ican (P,0.05) di e ence be ween he clones. S a s abo e he columns o ansgenic lines deno e signi ican (P,0.05) di e ence be ween ansgenic line o non- ansgenic clone. doi:10.1371/jou nal.pone.0114434.g003 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 16 / 29 con ained my ice in 3-a abinose no de ec ed in o he clones. The o al la onoid concen a ion as well as he o al con en o HPLC iden i ied phenolic compounds was highe (P,0.05) in clone A. Mo eo e , he concen a ion o soluble CTs and o al CTs was highes in clone A (Fig. 3B, S10 Table in S5 File). The lea con en s o cinnamic acid and que ce in de i a i es as well as insoluble CTs a ied signi ican ly (P,0.05) be ween clone A and P 4CL1a line A1 (Fig. 3B, S10 Table in S5 File). One cinnamic acid de i a i e was signi ican ly highe in line A5. Mo eo e , signi ican di e ence be ween clone A and line A5 was ound in he concen a ion o neolignan which was, in addi ion, signi ican ly educed in A1 line. Be ween clone E5382 and P 4CL1a line E5382/3, as well as be ween clone E5396 and line E5396/4, signi ican di e ences we e de ec ed in he concen a ion o chlo ogenic acid, cinnamic acid de i a i es, hype in and que ce in de i a i es (Fig. 3B, S10 Table in S5 File). Wounding caused only some changes in he phenolic compound concen a ions The e ec o mechanical wounding o phenolic compounds was s udied wi h lea samples collec ed 21 days a e ea men . In gene al, he wounding caused educ ion in he que ce in, kaemp e ol as well as o al la onoid concen a ions (Fig. 4, S11 Table in S5 File). No appa en end was de ec ed in he con en o cinnamic acid de i a i es, whe eas concen a ions o soluble CTs inc eased in all lines and clones apa o clone A (Fig. 4, S11 Table in S5 File). O he wise, he le els o phenolic compounds al e ed mo e due o he wounding in clones A and R han in clones E5382 and E5396, as only ew signi ican changes we e de ec ed in he concen a ion o indi idual compounds (S11 Table in S5 File). O clones A and R he la e had g ea e esponse ound in pa icula in he inc eased concen a ion o (+)-ca echin, neolignan, p o oca echuic acid, and soluble CTs (Fig. 4). The clones and co esponding ansgenic lines esponded simila ly o he wounding. Discussion The 4CL iso o ms a e able o use a a ie y o hyd oxycinnamic acids as subs a es, and hence, he ex en o gene amily expansion and subs a e di e si y has been ound o be connec ed [8]. In a ious plan species, he biosyn hesis o monolignols has been conside ed o be he main unc ion o he class I 4CL genes [9,14–20]. The class II 4CL genes ha e been sugges ed o unc ion, in addi ion o lignin biosyn hesis, in he syn hesis o la onoids [9,17–19,63]. In he p esen s udy, ou pu a i e sil e bi ch 4CL genes we e cloned and hei exp ession was moni o ed in s ems and lea es o sil e bi ches. The phylogene ic ML ee, econs uc ed using cds o cha ac e ized 4CL genes and 4CL-like ACS genes, was in acco dance wi h he gene al iew o class I and II membe s [13,18,64] as well as wi h he s udy o de Aze edo Souza e al. [23] p esen ing he e olu ion o 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 17 / 29 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 18 / 29 4CL-like ACS genes based on hei ansla ed nucleo ide sequences. The phylogene ic analysis sugges ed ha wo o he pu a i e 4CL genes o sil e bi ch belong o class I and one 4CL gene would belong o class II. The h ee 4CL genes, Bp4CL1,Bp4CL2, and Bp4CL4, we e exp essed in s ems o all s udied sil e bi ch clones. The 4CL-like ACSs a e a land plan -speci ic g oup o genes encoding adenyla e- o ming enzymes which con ain C- e minal consensus PTS1 pe oxisomal a ge sequence [23]. The 4CL-like ACS genes o P. ichoca pa (Pop ACS1,Pop ACS2), A abidopsis (A ACS6) and ice (OsACS1) we e shown o o m one o he i e conse ed clades o 4CL-like ACS sequences and we e de ec ed o exp ess in a ious o gans and issues o A abidopsis and P. ichoca pa [23]. In he p esen s udy, he phylogene ic analyses sugges ed ha Bp4CL3 would be 4CL-like ACS gene and u he mo e, would belong o he same clade wi h Pop ACS1, Pop ACS2,A ACS6 and OsACS1. The Bp4CL3 exp ession le els a ied conside ably among s udied sil e bi ch clones being, howe e , below de ec ion le el wi h used qPCR me hod in he lea es and s ems o clone A. The Bp4CL3 showed no esponse o mechanical wounding. Simila ly, he A ACS6 and Pop ACS2 we e shown o be un esponsi e o mechanical wounding as well as o simula ed he bi o y and he bi o y ea men s [23]. Howe e , wounding and he bi o y induced he exp ession o Pop ACS1 and 4CL-like ACS genes o o he clades indica ing ha some 4CL-like ACS genes also may unc ion in de ence p ocesses [23]. The an isense s a egy wi h 4CL genes has been success ully u ilized wi h se e al plan species o educe he lignin con en [16,20,25,28,29]. In he p esen s udy, sil e bi ch eli e clones A, E5382 and E5396 we e ans o med wi h he pRT99/ 35S-P 4CL1-a and i e sil e bi ch lines we e subsequen ly egene a ed. O hese A1, A5 and E5382/3 o igina ing om clones A and E5382, espec i ely, we e con i med o exp ess P 4CL1 unde s anda d g eenhouse condi ions. The P 4CL1a lines we e sho e han he non- ansgenic clones du ing he i s g owing season, howe e , a he end o hi d g owing season, he heigh s di e ed only be ween clone A and P 4CL1a line A1. Mo eo e , he g ow h o ansgenic sil e bi ch lines only exp essing np II (A2, E5396/4) was al e ed, possibly e lec ing he un a ou able inse ion si e o ansgene/s. Fu he mo e, he au umn lea senescence, ollowed du ing he end o i s g owing season, was delayed especially in he A2 line bu also in some ex en in lines A1 and A5 in compa ison wi h he clone A. The delay on he lea senescence was p obably connec ed o he di e ences in plan heigh s as he sho es plan s u ned he lea colou las and hence, could pa ly e lec shaded ligh condi ions [65]. The lea es o second g owing season we e o simila ood quali y o de olia o s among clones and lines Fig. 4. Wounding e ec s on he lea phenolics o sil e bi ch (Be ula pendula). The con en s o kaemp e ols (A, B), que ce in (C, D), and p-OH- cinnamic acid de i a i es (E, F), o al con en o HPLC iden i ied phenolics (G, H), soluble CT (I, J), o al CT (K, L), (+)-ca echin (M, N) and p o oca echuic acid (O, P) concen a ions in un ea ed (c) and mechanically wounded (w) lea es collec ed a day 0 and 21 d a e ea men in clones A and R and P 4CL1a lines A1 and A5 (A, C, E, G) and clones E5382 and E5396 and ansgenic lines E5382/3 and E5396/4 (B, D, F, H). Values a e mg/DW g mean ¡SE. doi:10.1371/jou nal.pone.0114434.g004 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 19 / 29 indica ing ha he ans o ma ion did no cause d as ic al e a ions in he lea chemis y o sil e bi ch lines. The P 4CL1 in an isense o ien a ion did no cause changes in he lignin con en o sil e bi ch lines and mo eo e , al e ed lignin composi ion was de ec ed only in he P 4CL1a line A1. The ela i e exp ession o P 4CL1 was g ea es in A1 line which, besides, showed highes copy numbe o in eg a ed P 4CL1a cons uc s. Voelke e al. [29] demons a ed wi h hyb id popla ha lignin quan i y and S/G a io can be modi ied by educing he 4CL1-1 (homolog o Pop 4CL3) le els a ound 60% and he 4CL2-2 (Pop 4CL5 homolog) le els a ound 95% o he exp ession le els in con ol popla . In he p esen s udy, he P 4CL1 sha ed g ea es simila i y wi h he cds sequence o Bp4CL1 and, indeed, signi ican al e a ion was ound in he Bp4CL1 exp ession o P 4CL1a A1 s ems. Howe e , he Bp4CL1 le els o A1 line we e s ill mo e han 60% o he con ol clone A when A ub was u ilized as he e e ence gene, possible explaining why he lignin con en o A1 line emained unchanged. Mo eo e , i is likely ha in P 4CL1a lines A5 and E5382/3, he exp ession le els o P 4CL1 we e no su icien o cause equi ed educ ion in he ansc ip le els o Bp4CL1 o cause changes in he lignin composi ion. Wounding has been shown o induce he exp ession o genes encoding enzymes o he gene al phenylp opanoid ou e leading o he gene a ion o monolignols essen ial o he wound sealing [19]. Thus, he phenylp opanoid lux should be di ec ed om he la onoid biosyn hesis ou e owa ds biosyn hesis o mono- lignols and, indeed, wounding has been shown o dec ease he exp ession o po en ial lignin biosyn hesis ep esso s [66,67]. In he p esen s udy, he exp ession o Bp4CL1 and Bp4CL2 was induced ansien ly 3 and 24 h a e wounding, espec i ely. On he con a y, he class I gene, Bp4CL4, was un esponsi e o wounding ea men . The induc ion o 4CL genes has been de ec ed by moni o ing he ansc ip le els and/o by p omo e analyses using po a o (Solanum ube osum)[ 14], A abidopsis [9,19], P. emuloides [68], ea (Camellia sinensis)[ 69], ice [64], Sal ia mil io hiza [70], and ke nel (Hibiscus cannabinus)[ 71]. The exp ession and p omo e s udies ha e demons a ed ha he egula ion o 4CLs is complex and includes elemen s, posi ioned bo h ups eam and also wi hin he cds, ac ing posi i ely and nega i ely [19] and, hus di e en 4CL amily membe s show dis inc exp ession pa e ns due o wounding [9,19,64]. In A abidopsis, he class I 4CL genes A 4CL1 and A 4CL2 showed biphasic induc ion wi h exp ession peaks 2.5 h and 48 h a e wounding [19]. The esponse o he A 4CL3 connec ed o la onoid biosyn hesis was i s educed and hen s eadily inc eased up o he 72 h a e wounding which ep esen ed he la es ime poin moni o ed [19]. When compa ed o he exp ession pa e ns o Bp4CL1 and Bp4CL2, in he p esen s udy, hey we e simila wi h he ea ly esponses o A 4CL1,A 4CL2 and A 4CL3 in A abidopsis as desc ibed by Sol ani e al. [19]. The geno ype o sil e bi ch has subs an ial in luence on he phenolic composi ion [72–75]. In he p esen s udy, soluble phenolic compounds a ied in all ou s udied sil e bi ch clones and di e ences we e de ec ed in bo h s ems and lea es in he le el o indi idual compounds, as well as compound g oups such as 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 20 / 29 cinnamic acids and phenolic glycosides. In P 4CL1a lines and clones signi ican di e ences we e de ec ed mos ly in he concen a ions o indi idual compounds, al hough, educed concen a ion o cinnamic acid de i a i es and phenolic glycosides we e de ec ed in indi idual P 4CL1a lines. In addi ion, he E5396/4 line exp essing only np II showed al e ed phenolic compound concen a ions in compa ison wi h he non- ansgenic clone E5396. This indica es ha , in addi ion o he abe an g ow h, he inse ion si e/s o ansgenes may ha e caused al e a ions o he phenylp opanoid pa hway which was, in addi ion, ound as changed ansc ip le els o sil e bi ch 4CL genes. The dec ease in he 4CL exp ession has led o al e ed lux o in e media es o he phenylp opanoid ou e which has been shown o inc ease he con en o cinnamic acid de i a i es [11,20,25,28] and o he phenolics [29,32]. In he p esen s udy, howe e , he P 4CL1a line A1 ha ing al e ed S/G a io, showed only inc ease in he (+)-ca echin con en o s ems and he insoluble CT con en o lea es. In gene al, he p oduc ion o phenolic compounds is induced in woody deciduous plan s upon damage, howe e , he esponse a ies acco ding o he s udied species, plan on ogeny, g ow h a e, and iming o he damage [7,76,77]. In he p esen s udy, he wounding caused only mino changes in he s udied soluble phenolic compounds examined o lea es collec ed 21 d a e wounding. As an o e all end he de i a i es o kaemp e ol and que ce in as well as o al concen a ion o la onoids we e educed in he inju ed lea es. Simila ly Muilenbu g e al. [78] ound educ ion in he con en o low molecula weigh phenolics in sil e bi ch and B.papy i e a and p oposed ha phenolics we e possibly u ilized o he p oduc ion o mo e complex polyphenols o polyme s such as lignin. In he p esen s udy, he wounding esponse a ied be ween clones: E5382 and E5396 esponded mildly whe eas clone R showed he s onges esponse, which was ound, o ins ance, as he ele a ed concen a ions o neolignan, p o oca echuic acid and (+)-ca echin, he la e o which can be u ilized as a p ecu so o CT syn hesis. Wounding has been epo ed o inc ease he CT concen a ions in P. emuloides [79] and in u leg ass (Thalassia es udinum)[80]. Mo eo e , MYB134, which egula es genes ela ed o he biosyn hesis o CTs, has been shown o be wound inducible [81]. Howe e , he induc ion o CT syn hesis upon he bi o y is species speci ic [7]. Fo ins ance, de olia ion o sil e bi ch did no cause induc ion in he lea CT le els [72] and wounding o sil e bi ch and B. papy i e a s ems caused educ ion in CTs [78]. Simila ly, lea damage ea men s o Que cus species and Ace opalus ssp. g ana ense [82–84] caused educ ion in he annin le els. In he p esen s udy, howe e , soluble CTs inc eased while insoluble CTs dec eased in he lea es o clone R. This, oge he wi h inc eased (+)-ca echin con en , sugges s ha CT accumula ion had possible no eached i s maximum in clone R. The 4CLs ha e been s udied ex ensi ely because o hei cen al ole in he gene al phenylp opanoid and monolignol biosyn hesis ou e leading o lignin wi h economic impo ance and a i al ole o he p ope unc ion o wood. Howe e , in he ligh o ecen indings [85–87], i seems ha he s udies on he 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 21 / 29 monolignol biosyn hesis ou e a e a om o e . In gene al, he indi idual 4CL genes ha e been conside ed o ha e plan pa speci ic exp ession pa e ns ela ed o de elopmen al and non-de elopmen al p ocesses. Howe e , he unc ion and egula ion o 4CLs has been ound o be mo e complex, o ins ance, Chen e al. [87] showed ha subuni s o wo P. ichoca pa 4CLs, 4CL3 and 4CL5, we e able o in e ac and o m he e o e ame ic p o ein complex which a ec s he di ec ion and a e o me aboli e luxes. In he p esen s udy we show ha sil e bi ch has a leas wo class I and one class II 4CL genes ha ing clone speci ic exp ession pa e ns as well as one 4CL-like ACS gene. The esul s indica e ha class I 4CL,Bp4CL1, unc ions in he monolignol biosyn hesis ou e as he ansc ip le els we e educed in he P 4CL1a line A1 ha ing al e ed S/G a io. The P 4CL1 did no cause d as ic al e a ions in he phenolics o s udied P 4CL1a, lines pe haps caused by insu icien ansc ip le els and/o de elopmen al s age o young plan s. The speci ic unc ions o sil e bi ch 4CLs emain o be sol ed. Howe e , Bp4CL1 could se e as a candida e o al e he lignin cha ac e is ics in sil e bi ch. Suppo ing In o ma ion S1 File. This ile con ains S1-S16 Figu es. S1 Figu e. A schema ic p esen a ion o he pRT99/35S-P 4CL1-a plasmid ec o used in he biolis ic ans o ma ion o sil e bi ch clones A, E5382 and E5396. S2 Figu e. The sca e plo s p esen ing CV% and concen a ion alues o s em samples. The CV% (SD Cq /mean Cq ) alues we e calcula ed om wo echnical eplica es o each u ilized p ime pai and he concen a ion alues we e gene a ed wi h Abs Quan /2nd De i a i e Max o All Samples Analysis o Ligh cycle 480 So wa e elease 1.5.0 SP3. A, A ub;B,PP2A; C, Bp4CL1;D,Bp4CL2;E,Bp4CL3;F,Bp4CL4;G,P 4CL1.S3 Figu e. The sca e plo s p esen ing CV% and concen a ion alues o lea samples. The CV% (SD Cq /mean Cq ) alues we e calcula ed om wo echnical eplica es o each u ilized p ime pai and he concen a ion alues we e gene a ed wi h Abs Quan / 2nd De i a i e Max o All Samples Analysis o Ligh cycle 480 So wa e elease 1.5.0 SP3. A, A ub;B,PP2A;C,Bp4CL1;D,Bp4CL2;E,Bp4CL3;F,Bp4CL4;G, P 4CL1.S4 Figu e. The Cq alues o A ub and PP2A in undamaged and wounded lea es. The Cq alues ep esen c ossing poin alues gene a ed wi h Abs Quan / 2nd De i a i e Max o All Samples Analysis o Ligh cycle 480 So wa e elease 1.5.0 SP3 o A ub (A) and PP2A (B) in non- ea ed lea es (c) and lea es collec ed immedia ely a e mechanical wounding (w0) and 1 (w1h), 3 (w3h), 12 (w12h), 24 (w24h), 72 (w3d) and 168 (w7d) hou s a e he wounding ea men . S5 Figu e. Sou he n blo analysis o egene a ed sil e bi ch lines. Lines A1, A2, A5 and E5382/3 we e ans o med wi h pRT99/35S-P 4CL1-a plasmid ec o and p obed o he p esence o he 35S-P 4CL1 and np II. A genomic DNA sample o 15 mg diges ed wi h BamHI ( igu es on he le ) o XbaI ( igu es on he igh ) was loaded o each lane. S6 Figu e. Sou he n blo analysis o egene a ed sil e bi ch line E5396/4. Line was ans o med wi h pRT99/35S-P 4CL1-a plasmid ec o and 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 22 / 29 p obed o he p esence o he np II. A genomic DNA sample o 15 mg diges ed wi h BamHI ( igu es on he le ) o XbaI ( igu es on he igh ) was loaded o each lane. S7 Figu e. No he n blo analysis o ansgenic sil e bi ch lines. RNA samples o 15 mg we e isola ed om he lea (L), phloem (P) and xylem (X) o clones A and E5382 and lines A1, A2, A5 and E5382/3. The p obes we e 1.1 and 0.8 kb o P 4CL1 and np II, espec i ely. S8 Figu e. The lea mo phology o P 4CL1a line E5382/3. Du ing he i s g owing season in he g eenhouse, he lea ma gins o P 4CL1a line E5382/3 (on he le ) di e ed in om clone E5382 (on he igh ). S9 Figu e. The au umn lea senescence o sil e bi ch clones and lines. The clone A, line A2, line A5, line E5382/3, and clone E5382 ( om le o igh ) a he end o i s g owing season a he g eenhouse o Finnish Fo es Resea ch Ins i u e Punkaha ju Uni (pho og aphed on he 6 h o Oc obe ). S10 Figu e. Alignmen o p edic ed amino acid sequence o sil e bi ch (Be ula pendula) pu a i e 4-couma a e:CoA ligase (Bp4CL1, KM099195). Bp4CL1 aligned wi h he p edic ed amino acid sequences o Be ula lumini e a (Bpl4CL1, AY792353), Be ula pla yphylla (Bl4CL, FJ410448), Glycine max (Gm4CL2, Glyma13g44950; Gm4CL6, Glyma15g00390), Maniho esculen a (Mes4CL2, cassa a4.1_005014m; Mes4CL3, cassa a4.1_005006m), Populus emuloides (P 4CL1, AF041049), and Populus ichoca pa (Pop 4CL3, g ail3.0100002702 LG I; Pop 4CL5, genesh4_pg.C_LG_III001773 LG III). S11 Figu e. Alignmen o p edic ed amino acid sequence o sil e bi ch (Be ula pendula) pu a i e 4-couma a e:CoA ligase (Bp4CL2, KM099196). Bp4CL2 aligned wi h he p edic ed amino acid sequences o A abidopsis haliana (A 4CL3, A 1g65060), Rubus idaeus (Ri4CL3, AAF91308), Glycine max (Gm4CL3, NM_001250341), Maniho esculen a (Mes4CL1, cassa- a4.1_004658m; Mes4CL4, cassa a4.1_004136m), Populus emuloides (P 4CL2, AF041050), and Populus ichoca pa (Pop 4CL4, g ail3.0099003002 LG IX). S12 Figu e. Alignmen o p edic ed amino acid sequence o sil e bi ch (Be ula pendula) pu a i e 4CL-like acyl-CoA syn he ase (ACS) (Bp4CL3, KM099197). Bp4CL3 aligned wi h he p edic ed amino acid sequences o A abidopsis haliana (A ACS6, A 4g05160), O yza sa i a (OsACS1, Os03g05780) and Populus ichoca pa (Pop ACS1, eugene3.01230068; Pop ACS2, es EXT_ genesh1_pg_ 1.C_LG_IV0024). S13 Figu e. Alignmen o p edic ed amino acid sequence o sil e bi ch (Be ula pendula) pu a i e 4-couma a e:CoA ligase (Bp4CL4, KM099198). Bp4CL4 wi h he p edic ed amino acid sequences o Rubus idaeus (Ri4CL2, AAF91309), Glycine max (Gm4CL7, Glyma17g07170; Gm4CL8, Glyma17g07180; Gm4CL1, Glyma17g07190; Gm4CL9, Glyma13g01080), and Populus ichoca pa (Pop 4CL1, es Ex _ genesh4_pg.C_1210004 sca old3; Pop 4CL2, gw1.XVIII.2818.1 LG_XVIII). S14 Figu e. Rela i e exp ession o P 4CL1 in s ems and lea es o P 4CL1a lines A1, A5 and E5282/3. Values ep esen means and s anda d de ia ions calcula ed using PP2A gene as e e ence gene (n53–14). S15 Figu e. Rela i e exp ession o Bp4CL1-4genes in mechanically wounded sil e bi ch (Be ula pendula) lea es. The Bp4CL1 (A, C) and Bp4CL2 (B, D) exp ession in lea es collec ed immedia ely (w0) and 1 (w1), 3 (w3), 12 (w12), 24 (w24), 72 (w3d), and 162 (w7d) h a e mechanical wounding o clones A, E5382, E5396 and 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 23 / 29 R and ansgenic lines A1, A5, E5382/3 and E5396/4. Values ep esen means and s anda d e o s calcula ed om he a ge / e ence a ios. A and C, PP2A was used as he e e ence gene; B and D, A ub was used as he e e ence gene. S16 Figu e. Rela i e exp ession o Bp4CL3 (A, B), and Bp4CL4 (C, D) in wounded lea es. The lea es we e collec ed immedia ely (w0) and 1, 3, 12, 24, 48, and 162 h a e mechanical wounding o sil e bi ch (Be ula pendula) clones A and R and ansgenic lines A1 and A5 (A, C) and clones E5382 and E5396 and ansgenic lines E5382 and E5396 (B, D). Values ep esen means and s anda d e o s calcula ed using he mean o con ol lea es as no malize wi hin each line/clone and PP2A as e e ence. doi:10.1371/jou nal.pone.0114434.s001 (PDF) S2 File. This ile con ains S1-S2 Tables. S1 Table. The p ime s used in he p oduc ion he p obes, subcloning o cds o pu a i e Bp4CL1-4 and eal- ime RT- PCR. S2 Table. The pe o mance o A ub and PP2A p ime s used in he ampli ica ion o e e ence genes. doi:10.1371/jou nal.pone.0114434.s002 (PDF) S3 File. This ile con ains S3–S5 Tables. S3 Table. The Cq (c ossing poin ) and concen a ion alues o s ems. S4 Table. The Cq (c ossing poin ) and concen a ion alues o lea es. S5 Table. The 4CL and 4CL-like ACS sequences used in he gene a ion o ML ee. doi:10.1371/jou nal.pone.0114434.s003 (XLS) S4 File. This ile con ains S6–S8 Tables. S6 Table. The g ow h cha ac e is ics o sil e bi ch non- ansgenic clones and ansgenic lines. S7 Table. The phenology o au umn lea senescence a he end o i s g owing season. S8 Table. Rela i e g ow h a es (mg/d) o lepidop e an la ae. The lea es o sil e bi ch clones (A and E5382) and lines (A1, A2, A5 and E5382/3) we e o e ed o he la ae o Ae halu a punc ula a,Cleo a cinc a ia,Epi i a au umna a, and O hosia go hica. doi:10.1371/jou nal.pone.0114434.s004 (PDF) S5 File. This ile con ains S9–S11 Tables. S9 Table. Concen a ions o phenolic compounds in sil e bi ch s ems. S10 Table. Concen a ions o phenolic compounds in sil e bi ch lea es. S11 Table. Concen a ions o phenolic compounds in sil e bi ch lea es 21 d a e wounding. doi:10.1371/jou nal.pone.0114434.s005 (XLS) Acknowledgmen s The au ho s a e g a e ul o he s a o he Finnish Fo es Resea ch Ins i u e Punkaha ju Uni and Bo anical Ga dens o he Uni e si y o Oulu o hei excellen assis ance. I meli Luo ula, Ta ja To ¨ ma ¨nen, Susanna Ha ¨ggman and Taina Uusi alo a e acknowledged o hei kind assis ance. 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 24 / 29 Au ho Con ibu ions Concei ed and designed he expe imen s: TA HH SS HT TY. Pe o med he expe imen s: TA TH TL SS HT TY. Analyzed he da a: TA TH TL PS SS TY. Con ibu ed eagen s/ma e ials/analysis ools: PS VC RJT. W o e he pape : HH TH RJT SS HT TY. Re e ences 1. Bue CS, Imin N, Djo dje ic MA (2010) Fla onoids: New oles o old molecules. J In eg Plan Biol 52: 98–111. 2. Boeckle GA, Ge shenzon J, Unsicke SB (2011) Phenolic glycosides o he Salicaceae and hei ole as an i-he bi o e de enses. 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Plan Mol Biol 51: 327–340. 4CL Exp ession in Sil e Bi ch PLOS ONE | DOI:10.1371/jou nal.pone.0114434 Decembe 11, 2014 25 / 29