Characterization of cytokinin signaling and homeostasis gene families in two hardwood tree species: Populus trichocarpa and Prunus persica

RESEARCH ARTICLE Open Access Cha ac e iza ion o cy okinin signaling and homeos asis gene amilies in wo ha dwood ee species: Populus ichoca pa and P unus pe sica Juha Immanen 1 , Kaisa Nieminen 2 , Héc o Duchens Sil a 3 , Fe nanda Rod íguez Rojas 4,5 , Lee A Meisel 4,5 , He man Sil a 3 , Vic o A Albe 6 , To gei R H ids en 7,8 and Ykä Hela iu a 1* Abs ac Backg ound: Th ough he di e si y o cy okinin egula ed p ocesses, his phy oho mone has a p o ound impac on plan g ow h and de elopmen . Cy okinin signaling is in ol ed in he con ol o apical and la e al me is em ac i i y, b anching pa e n o he shoo , and lea senescence. These p ocesses in luence se e al ai s, including he s em diame e , shoo a chi ec u e, and pe ennial li e cycle, which de ine he de elopmen o woody plan s. To acili a e esea ch abou he ole o cy okinin in egula ion o woody plan de elopmen , we ha e iden i ied genes associa ed wi h cy okinin signaling and homeos asis pa hways om wo ha dwood ee species. Resul s: Taking ad an age o he sequenced black co onwood (Populus ichoca pa) and peach (P unus pe sica) genomes, we ha e compiled a comp ehensi e lis o genes in ol ed in hese pa hways. We iden i ied genes belonging o he six amilies o cy okinin oxidases (CKXs), isopen enyl ans e ases (IPTs), LONELY GUY genes (LOGs), wo-componen ecep o s, his idine con aining phospho ansmi e s (HP s), and esponse egula o s (RRs). All oge he 85 Populus and 45 P unus genes we e iden i ied, and compa ed o hei A abidopsis o hologs h ough phylogene ic analyses. Conclusions: In gene al, when compa ed o A abidopsis, di e ences in gene amily s uc u e we e o en seen in only one o he wo ee species. Howe e , one class o genes associa ed wi h cy okinin signal ansduc ion, he CKI1-like amily o wo-componen his idine kinases, was la ge in bo h Populus and P unus han in A abidopsis. Keywo ds: Cy okinin signaling, Cy okinin homeos asis, Populus ichoca pa, Black co onwood, P unus pe sica, Peach Backg ound Cy okinin signaling con ibu es o he egula ion o mul- iple undamen al p ocesses ac i e in plan de elopmen . These include cell di ision, me is em main enance, shoo ini ia ion and g ow h, ascula pa e ning, lowe and seed de elopmen , nu ien up ake, chlo oplas di e en ia ion and ligh pe cep ion [1-3]. Addi ionally, his ho mone plays a ole in egula ing se e al de elopmen al p og ams de ining he li e o pe ennial woody plan s, including he ac i i y o ascula cambium, b anching pa e n o he shoo , and he onse o lea senescence. The long li e span and ex ensi e adial g ow h con ibu e o he la ge size and massi e amoun o wood p esen in a ee, c ea ing a s a k con as o he much smalle he baceous annuals. Howe e , only ew s udies ha e hus a been published abou he ole o cy okinin in he egula ion o woody plan de elopmen . To acili a e his esea ch, we a e now p esen ing a comp ehensi e desc ip ion o cy okinin sig- naling and homeos asis gene amilies in wo ha dwood ee species: Populus ichoca pa and P unus pe sica. Gene iden i ica ion in ee genomes was based on hom- ology wi h A abidopsis genes, as cy okinin homeos asis and signal ansduc ion pa hways ha e been ex ensi ely s udied and well-cha ac e ized in his species [1-3]. S uc u ally, cy okinins a e adenine de i a i es; based on side chain iden i y hey can be classi ied in o ou * Co espondence: [email p o ec ed] 1 Ins i u e o Bio echnology and Depa men o Biosciences, Uni e si y o Helsinki, FI-00014 Helsinki, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2013 Immanen e al.; licensee BioMed Cen al L d. 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 (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Immanen e al. BMC Genomics 2013, 14:885 h p://www.biomedcen al.com/1471-2164/14/885 g oups ep esen ing isopen enyladenine (iP), ans-zea in ( Z), cis-zea in, and a oma ic cy okinins. iP and Z a e he bioac i e o ms o his ho mone, o which plan s espond h ough a mul is ep wo-componen his idine-aspa a e (His-Asp-His-Asp) phospho elay sys em [4-6]. The phos- pho elay is ini ia ed when a cy okinin ligand binds o a his idine kinase ecep o , which igge s au ophospho yl- a ion o a His esidue. A e an in amolecula ans e o he phospho yl o an Asp esidue, i will be ans e ed o a His in a cy osolic his idine phospho ans e (HP ) p o- ein. The HP s p o ide a mobile connec ion be ween he cy osol and nucleus; hey con inuously cycle be ween hese wo compa men s. In he nucleus, he HP ans e s he phospho yl on o an Asp in a phospho-accep ing e- sponse egula o (RRs). RRs can be classi ied in o se e al di e en ypes acco ding o hei s uc u e and unc ion. Type-B RRs, which belong o he Myb- ansc ip ion ac- o s, ac i a e he ansc ip ion o cy okinin p ima y e- sponse genes. Among hem a e he ype-A RRs, which a e in ol ed in a nega i e eedback mechanism ha helps o ine- une he unc ion o cy okinin signaling pa hway. Type-A RRs ep ess ac i i y o ype-B RRs [4,7] and a e s abilized by HP media ed phospho yla ion (To e al. [8]). Adding u he lexibili y o he signaling pa hway, many o i s componen s a e capable o o ming bo h homo- and he e odime s [9-13]. Di e en combina ions o he wo- componen elemen s p esumably add di e si y in o he p ocess and ou come o he phospho elay. Cy okinin signaling ep esen s an ancien ho monal pa hway. All o i s componen s a e al eady p esen in he genome o moss Physcomi ella pa ens [14,15], indica ing ha he cy okinin phospho elay was al eady unc ional p io o he de elopmen o a well-de ined plan ascula- u e. As compa ed o he moss, he cy okinin signaling pa hway has, howe e , become mo e di e se du ing he e olu ion o land plan s. The numbe o membe s in mos cy okinin signaling gene amilies is much highe in he ge- nomes o ascula plan s han in Physcomi ella [14,15]. In gene al, he dynamic na u e o plan genomes has in lu- enced he e olu ion o all gene amilies in ascula plan s. All angiospe m lineages ha e unde gone eoccu ing gen- ome duplica ions, indica ing ha polyploidiza ion con e s a i ness ad an age o plan species. Each ad en o a whole genome duplica ion is subsequen ly ollowed by a g adual gene loss; his ediploidiza ion ul ima ely p o- mo es a new duplica ion, allowing he p ocess o epea in a cyclical manne [16]. To s udy he s uc u e o cy okinin signaling and homeos asis genes amilies in woody plan s, we sough o cha ac e ize and compa e hem be ween wo ha d- wood ee species. Fo he i s species in ou phylogen- e ic s udy, we chose he mos common model ee o molecula biology: Populus ichoca pa, black co on- wood. Populus is a as g owing a dioecious ee, which can each ep oduc i e ma u i y in ou o six yea s. Populus ees p o ide a wood sou ce o he pulp and pape indus y and ha e he po en ial o be de eloped in o a bio uel eeds ock [17]. P. ichoca pa has a ela- i ely small diploid (2n = 38) genome wi h he haploid size o 485 Mbp. The i s e sion o genome assembly was published in 2006 by Tuskan e al. [18]. Due o he challenges o genome assembly in a highly he e ozygous ee species, only he cu en , hi d genome assembly o P. ichoca pa, has been able o esol e a la ge numbe o eads ha we e p e iously published as unassembled sca olds. Speci ic loci iden i ies ha e only ecen ly been assigned o all p edic ed genes. Thanks o hese im- p o emen s, we ha e now o he i s ime been able o eliably eco e a comple e se o cy okinin signaling and homeos asis genes om a ee species. Acco dingly, we will discuss how ou analysis di e s om p e iously published epo s o P. ichoca pa cy okinin signaling genes [14,19,20]. The second ha dwood ee species used in his s udy is he economically impo an ui ee peach, P unus pe sica. In e ms o cul i a ed su ace a ea, P. pe sica is he hi d mos impo an empe a e ui c op. Addi ion- ally, i is a membe o he economically impo an Rosa- ceae amily, which includes impo an c ops such as peaches, apples, pea s, che ies, plums, ap ico s, s aw- be ies, almonds, and oses. An in e na ional e o has led o he genome sequencing and de elopmen o P u- nus pe sica as a genomic model o he Rosaceae amily [21-23]. This ha dwood ee is a sel -pollina ing diploid (2n = 16), wi h a sho ju enile pe iod (2–3 yea s) and a genome size o 265 Mbp [22,23]. Cu en ly only a li le is known abou he ole o cy oki- nin signaling in he egula ion o ee o ui de elopmen in Rosaceae. The a ailable da a indica es ha cy okinins a e impo an o ui de elopmen : high ho mone le els ha e been measu ed in g owing peach ui s [24]. I has also been demons a ed ha exogenous applica ion o cy okinin on swee che y ui s signi ican ly inc eases ui size and weigh [25]. Addi ionally, cy okinin ea ed ui s showed inc eased ui i mness, inc eased ui sol- uble solid concen a ions and a delay in exoca p colo - a ion [25]. Simila esul s ha e also been seen in apples and pea s ha ha e been ea ed exogenously wi h cy oki- nin [26-29]. Taking oge he , hese obse a ions indica e ha he cy okinin signaling and homeos asis pa hways can p o ide candida e genes o he b eeding o as g owing and high quali y Rosaceae ui s. The hi d species chosen o ou s udy is he mos common he baceous model plan , A abidopsis haliana. A abidopsis p o ides an excellen e e ence genome, as i s cy okinin homeos asis and signal ansduc ion pa h- ways ha e been cha ac e ized in de ail [1-3]. In addi ion o he con as be ween he woody pe ennial e sus Immanen e al. BMC Genomics 2013, 14:885 Page 2 o 12 h p://www.biomedcen al.com/1471-2164/14/885 he baceous annual li e cycles, he selec ed h ee model species di e in hei ep oduc i e s a egies. Bo h P unus and A abidopsis ha e he maph odi ic lowe s, whe eas Populus is a dioecious ee whose genomic sequence was de i ed om a emale plan [18]. All h ee model species belong o he osid clade o angiospe m plan s. Populus (Malpighiales) and P unus (Rosales) belong o he eu osids I subclade (Fabidae), whe eas A abidopsis (B assicales) belongs o he eu osids II (Mal idae) [30]. They display di e se genome duplica- ion his o ies: since hei las common ances o , Populus lineage has unde gone one whole genome duplica ion, A abidopis wo, and P unus none [18,31,32]. Based on he genome duplica ion his o y and numbe o synonymous nucleo ide subs i u ions, he molecula -clock a e has been calcula ed o be as e in A abidopsis han in Populus [33]. Due o he genome duplica ion his o y and gene e o- lu ion a e, he Populus genome has on a e age 1.5 o ho- logs o each A abidopsis gene [18], and P unus 0.85 [34] (h p://www. osaceae.o g/p ojec s/peach_genome/ 1.0/ homology). The di e ences in he cy okinin signaling and homeos asis ela ed gene amily sizes a e consis en wi h he gene al genomic ends. We iden i ied a o al o 85 genes om he Populus ichoca pa genome and 45 genes om P unus pe sica, as compa ed o he 60 A abidopsis genes. The gene amily s uc u es be ween he wo ee species and A abidopsis we e compa ed h ough phylo- gene ic analyses. Me hods Sequence alignmen s Populus and P unus homologues o A abidopsis genes we e iden i ied by sea ching he Populus ichoca pa genome da abase e sions 1.1 and 3.0 using a ious bio- in o ma ic ools and da abases a ailable ia he Populus genome po al (h p://genome.jgi-ps .o g/Pop 1_1/Pop 1_ 1.home.h ml; h p://www.phy ozome.ne /sea ch.php), and he P unus pe sica genome e sion 1 [23], using da abases a ailable ia The Genome Po al o he Depa men o Ene gy Join Genome Ins i u e [35] (h p://genome.jgi- ps .o g/Pop 1_1/Pop 1_1.home.h ml), and Phy ozome po al [36] (h p://www.phy ozome.ne /sea ch.php?o g= O g_P ichoca pa_ 3.0; h p://www.phy ozome.ne /sea ch. php?me hod=O g_Ppe sica). The bioin o ma ics ools included BLAST sea ches, Gene On ology (GO), Kyo o Encyclopedia o Genes and Genomes (KEGG), EuKa yo ic O hologous G oups Da abase (KOG) and o holog inde . A abidopsis sequences we e iden i ied using The A abidopsis In o ma ion Resou ce (TAIR). Amino acid sequences we e aligned using Clus alW ollowed by manual adjus men s when needed [37]. The bes bidi ec ional hi (BBH) me hod was used as he i s app oach o de e mine o hologous pai s o he cy okinin signaling and esponse genes in P unus as desc ibed by [38]. P o ein sequences we e aligned using Jal iewand Clus alW2 [39] ollowed by manual adjus men s whe e needed. The esul ing alignmen was p ecisely back- ansla ed o yield a da a ma ix o he co esponding nucleo ide sequences. Gene models, ansc ip IDs and physical loci o Populus genes used in cons uc ion o phylogene ic ees can be ound in Addi ional ile 1: Table S1. Gene models and EST suppo o P unus genes can be ound in Addi ional ile 2: Table S2 (physical loci o he genes a e no a ailable in he cu en 1.0 e sion o he genome), and TAIR gene numbe s o A abidopsis genes in Addi ional ile 3: Table S3. Phylogene ic anno a ion We used a maximum likelihood sea ch s a egy on amino acid alignmen s o in es iga e o hologs and pa alogs in he cy okinin signaling and homeos asis gene amilies. Se- quences we e aligned using MUSCLE wi h de aul se ings [40]. A single mos op imal ee o each da a se was compu ed using he RaxML BlackBox web se e (h p:// embne . i al-i .ch/ axml-bb/) unning RaxML e sion 7.2.8 [41]. De aul se ings we e used wi h he WAG model o molecula e olu ion including a gamma pa am- e e . One hund ed boo s ap samples we e gene a ed o assess suppo o he in e ed ela ionships. Local boo - s ap alues (in pe cen ages) a e indica ed o b anches wi h ≥50% suppo . Resul s and discussion To cha ac e ize he gene ic componen s o cy okinin sig- naling and homeos asis pa hways om Populus and P u- nus, we iden i ied genes belonging o he six amilies o cy okinin oxidases (CKXs), isopen enyl ans e ases (IPTs), LONELY GUY genes (LOGs), wo-componen ecep o s, his idine con aining phospho ansmi e s (HP s), and ype-B, ype-A, and ype-C esponse egula o s (RRs). Below we will b ie ly summa ize wha is known abou each gene amily in A abidopsis, a e which we desc ibe hem in ou wo ee species. Cy okinin oxidases Cy okinin oxidase/dehyd ogenases (CKXs) a e majo en- zymes esponsible o cy okinin ca abolism [42,43]. CKX p o eins sha e low sequence homology; he only con- se ed ea u es a e an oxido educ ase FAD-binding do- main and a ew sho consensus mo i s [44]. The Populus genome con ains eigh , P unus six and A abidopsis se en CKX genes (Figu e 1, Addi ional ile 4: Figu e S1). The s uc u e o he gene amily is well conse ed be ween all h ee species (Figu e 1). Isopen enyl ans e ases (IPTs) A majo s ep in cy okinin biosyn hesis is ca alyzed by ATP/ADP isopen enyl ans e ases (IPTs), which a e Immanen e al. BMC Genomics 2013, 14:885 Page 3 o 12 h p://www.biomedcen al.com/1471-2164/14/885 esponsible o mos o he iP and Z- ype cy okinin bio- syn hesis [45-49]. They belong o he IPT gene amily o- ge he wi h RNA IPTs, which a e esponsible o he biosyn hesis o cZ- ype cy okinins [48]. Bo h Populus and A abidopsis genomes con ain nine membe s o he IPT amily, whe eas P unus has se en (Figu e 2, Addi ional ile 5: Figu e S2). Bo h ee species ha e one o holog o each o he wo A abidopsis RNA IPT genes (IPT2 and IPT9) (Figu e 2). The s uc u e o his gene amily is o he wise ela i ely conse ed be ween he h ee plan species. LONELY GUY (LOG) genes Cy okinin concen a ion is locally egula ed h ough he ac i i y o LONELY GUY (LOG) enzymes, which con- e conjuga ed cy okinin nucleo ides in o hei bioac i e nucleobase o ms [50,51]. These enzymes a e impo an egula o s o shoo and oo apical me is em ac i i y [50-53]. The ac ion o LOGs enables a plan o sepa a e and de ine he exac si e and ime o cy okinin ac i a- ion, and espec i ely i s pe cep ion, apa om ha o i s biosyn hesis. The Populus genome con ains 13 genes Figu e 1 Un oo ed maximum likelihood ee o Populus ichoca pa (P ), P unus pe sica (Pp) and A abidopsis cy okinin oxidase/dehyd ogenase (CKX) genes coding o enzymes in ol ed in cy okinin ca abolism. The ee is based on a deduced amino acid (431 aa) sequence alignmen (Addi ional ile 4: Figu e S1). Suppo o each clade is gi en as ≥50% o boo s ap pseudo eplica es. Figu e 2 Un oo ed maximum likelihood ee o Populus (P ), P unus (Pp) and A abidopsis isopen enyl ans e ase (IPT) genes, which encode cy okinin biosyn he ic enzymes. The ee is based on a deduced amino acid (282 aa) sequence alignmen (Addi ional ile 5: Figu e S2). Suppo o each clade is gi en as ≥50% o boo s ap pseudo eplica es. Immanen e al. BMC Genomics 2013, 14:885 Page 4 o 12 h p://www.biomedcen al.com/1471-2164/14/885 coding o cy okinin ac i a ing LOG enzymes, P unus has se en and A abidopsis nine (Figu e 3, Addi ional ile 6: Figu e S3). The numbe o o hologs appea s o ha e mul iplied in one clade in he Populus lineage. This species has ou o hologs(P LOG5a-d)o A abidopisA LOG5, whe eas P unus has only one (PpLOG5)(Figu e3). Two-componen ecep o s The ini ial pe cep ion o bioac i e cy okinins akes place h ough CRE1-like wo-componen his idine kinase ecep- o s, which belong o he supe amily o wo-componen egula o s [5,54]. In addi ion o he cy okinin ecep o s, his amily con ains a wo-componen his idine kinase CKI1 ha is able o ac i a e he cy okinin phospho elay [55], i e e hylene ecep o s (ETR1, ETR2, ERS1, ERS2 and EIN4), i e phy och omes (PHYA-E), one pu a i e osmosenso (A HK1), and a his idine kinase (CKI2/AHK5) associa ed wi h e hylene and ABA signaling [56-58]. Ou ocus will be on he CRE1- and CKI1-like sub am- ilies ha a e known o pa icipa e in cy okinin signaling phospho elay. A abidopsis has h ee cy okinin ecep o s: CRE1/WOL/ AHK4, AHK2 and AHK3 [5,54,59]. These ecep o s ha e a cy okinin binding CHASE domain, ansmemb ane do- mains, a His kinase domain and a ecei e domain which con ains he phospho-accep ing Asp. They sha e o e lap- ping unc ions: single null mu an s do no ha e no able pheno ypes, whe eas he iple mu an is a se e ely dwa ed and in e ile plan [54]. One o he h ee ecep o s, CRE1, has bo h kinase and phospha ase ac i i y: upon binding cy okinin i phospho - yla es HP s, whe eas in he absence o he ho mone i in- s ead dephospho yla es hem [6]. I s phospha ase ac i i y helps o quickly inac i a e he phospho elay when he cy okinin le els dec ease. In addi ion o he h ee canon- ical ecep o s, A abidopsis has a ou h wo-componen his idine kinase, CKI1, which is capable o inducing cy o- kinin esponses [55]. This kinase can ini ia e he phos- pho elay, bu independen ly o cy okinin [6,60-62]. As i is missing he cy okinin binding CHASE domain, i does no ep esen a ue cy okinin ecep o . Fu he in con as o he CRE1-like ecep o s, which a e mainly loca ed a he endoplasmic e iculum [11,63], CKI1 appea s o be p esen a he plasma memb ane [4,64]. The Populus genome con ains i e cy okinin ecep o genes (P CRE1a,P CRE1b,P HK2,P HK3a and P HK3b) [19], and P unus h ee (PpCRE1,PpHK2,PpHK3), all o hologous o he h ee A abidopsis CRE1-like ecep o s (Figu e 4, Addi ional ile 7: Figu e S4). In con as , bo h ee species ha e h ee o hologs o CKI1 (P CKI1a-c; PpCKI1a-c), a single copy gene in A abidopsis (Figu e 4, Addi ional ile 7: Figu e S4). The signi ican (3- old) ex- pansion o he CKI1 gene amily appea s o be speci ic o he Populus and P unus lineages, as bo h soybean [65] and ice [66] a e simila o A abidiopsis, ha ing only one o holog o his gene. CKI1 is known o be essen ial o e- male game ophy e de elopmen [67], and in e es ingly, has also been epo ed o egula e ascula de elopmen in A abidopsis in lo escence s em. In he s udy by Hejá ko e al. [64], CKI1 exp ession was de ec ed in ascula is- sues, and i s o e -exp ession inc eased he numbe o as- cula cambial cells in ascula bundles. Acco dingly, he numbe o cambial cells was educed in RNAi lines whe e he CKI1 exp ession le el was down- egula ed [64]. The e o e, CKI1 appea s o ha e a s imula o y ole in egula ion o ascula cell p oli e a ion in A abidopsis. Figu e 3 Un oo ed maximum likelihood ee o Populus (P ), P unus (Pp) and A abidopsis LONELY GUY (LOG) genes. LOGs con e conjuga ed cy okinins in o hei bioac i e o ms. The ee is based on a deduced amino acid (198 aa) sequence alignmen (Addi ional ile 6: Figu e S3). Suppo o each clade is gi en as ≥50% o boo s ap pseudo eplica es. Immanen e al. BMC Genomics 2013, 14:885 Page 5 o 12 h p://www.biomedcen al.com/1471-2164/14/885 His idine con aining phospho ansmi e s Upon binding cy okinin, he CRE1-like ecep o s ini ia e phospho yla ion o his idine con aining phospho ansmi - e s (HP s), which a e con inuously cycling be ween cy o- sol and nucleus [12]. This mo emen enables he ans e o phospho yl g oups om he memb ane-localized ecep- o s o he nuclea -localized esponse egula o s. The HP s a e cha ac e ized by a sho mo i , HQxKGSSxS, which con ains a conse ed phospho-accep ing His esi- due (Addi ional ile 8: Figu e S5) [68]. In A abidopsis, i e membe s o he gene amily (AHP1-5) con ain his canon- ical consensus mo i [69-72]. They sha e pa ially edun- dan unc ions since highe -o de null mu an s, ha a e lacking mul iple genes om he same gene amily, display a p og essi ely educed sensi i i y o cy okinin [73]. In con as o he i e canonical membe s, wo A abidopsis HP genes, AHP6 and AHP-like (A 4g04402), con ain an a ypical mo i lacking he conse ed His esidue [71]. AHP6 has an inhibi o y ole on he cy okinin phospho e- lay, and has been classi ied as a pseudo HP [74]. AHP6 nega i ely in e e es wi h he phospho elay, po en ially by compe ing wi h he o he AHPs o in e ac ion wi h phos- ho yla ed ecep o s. In A abidopsis oo s, he exp ession o AHP6 p omo es di e en ia ion o p o oxylem, he i s xylem cell ype ha o ms in a de eloping ascula u e [74]. The nega i e unc ion o AHP6 con ibu es o he gene a ion o dis inc and well-de ined domains o low cy okinin signaling. The unc ion and exp ession pa e n o he AHP-like gene is no known. All oge he 14 HP -encoding genes we e iden i ied in he new Populus genome assembly (Figu e 5, Addi ional ile 8: Figu e S5); ou mo e han we e epo ed by Pils and Heyl [14]. Nine HP s we e iden i ied in P unus,ascom- pa ed o he se en in A abidopsis (Figu e 5, Addi ional ile 8: Figu e S5). The gene amily s uc u e is ela i ely di - e en be ween he ee species and A abidopsis (Figu e 5). The ees ha e one clade (P HP8a,P HP8b and PpHP8) wi h no e iden A abidopsis o hologs, and Populus has one mo e (P HP1a and P HP1b) wi hou ei he an A abidopsis o P unus o holog. Two Populus (P HP6a, P HP6b)andoneP unus HP (PpHP6) a e o hologous o he A abidopsis pseudo HP AHP6, and acco dingly lack he phospho-accep ing His esidue (Addi ional ile 8: Figu e S5). One Populus (P HP-like)andoneP unus gene (P HP-like) con ain non-canonical consensus mo- i s lacking he conse ed his idines (Addi ional ile 8: Figu e S5); i is no known i hese p o eins pa icipa e in he phospho elay. In e es ingly, bo h Populus and P unus ha e ou o ho- logs o a single A abidopsis gene, AHP4. Howe e , his is he case also in wo monoco species; ice and maize, which bo h ha e h ee o hologs o his gene [73,75,76]. Compa ed o o he species, i appea s ha A abidopsis has los i s AHP4 homologs du ing e olu ion. Somewha su p isingly, despi e he loss o hese po en ially edundan genes, he pheno ype o an A abidopsis AHP4 null mu an is no e y s iking. In A abidopsis, AHP4 is p ima ily exp essed in young lowe s, and he null mu an shows sligh ly mo e seconda y cell wall hickening a some Figu e 4 Un oo ed maximum likelihood ee o Populus (P ), P unus (Pp) and A abidopsis CRE1-andCKI1-like wo-componen his idine kinase genes. CRE1-like genes encode cy okinin ecep o s. In con as , CKI1 does no ep esen a ue ecep o : despi e being able o ac i a e cy okinin phopho elay, i is unable o bind cy okinin. All he o he membe s o A abidopsis wo-componen ecep o amily, which ha e no known ole in cy okinin signaling, a e also included. They include e hylene ecep o s (ETR1, ETR2, ERS1, ERS2 and EIN4), phy och omes (PHYA-E), a pu a i e osmosenso (A HK1), and a his idine kinase (CKI2/AHK5) associa ed wi h e hylene and ABA signaling. The ee is based on a deduced amino acid (113 aa) sequence alignmen (Addi ional ile 7: Figu e S4). Suppo o each clade is gi en as ≥50% o boo s ap pseudo eplica es. Immanen e al. BMC Genomics 2013, 14:885 Page 6 o 12 h p://www.biomedcen al.com/1471-2164/14/885 an he issues; ye i s e ili y is no impai ed [77]. P esum- ably he AHP4 unc ion is s ill edundan wi h he o he A abidopsis HP p o eins. One Populus AHP4 o holog (P HP4b) is missing he conse ed phospho-accep ing His esidue. This e lec s he si ua ion in monoco s, whe e all h ee ice AHP4 o hologs [73], and wo om he h ee maize o hologs, also lack he conse ed his idine [76]. These genes appea o ha e e ol ed in o pseudo- esponse egula o s wi h a po en ially inhibi o y ole on he cy oki- nin signaling phospho elay. Response egula o s Response egula o s (RRs) ep esen he inal componen s o he cy okinin signaling phospho elay. The common ea- u e o all RRs is a ecei e domain, which con ains he phospho-accep ing Asp esidue as pa o he co e se- quence DD-D-K (Asp Asp-Asp-Lys) [56,57]. The RRs can be classi ied in o ou sub amilies: A- ype RRs wi h he e- cei e domain; B- ype RRs wi h he ecei e domain used o a DNA-binding (GARP) sequence; C- ype RRs, which despi e an a ypical amino acid sequence o hei ecei e domain, s ill con ain he phospho-accep ing Asp esidue; and pseudo RRs lacking he conse ed Asp in hei e- cei e domain. Membe s o he ype-A, -B and -C RR sub- amilies pa icipa e in he cy okinin signaling phosho elay [56,57,78]. The pseudo RRs a e, ins ead o cy okinin sig- naling, known o unc ion in he egula ion o ligh e- sponses, including ci cadian hy hms [79-81]. We will no discuss hem in his a icle. Type-B RRs Type-B RRs a e DNA-binding ansc ip ional egula o s ha posi i ely media e cy okinin esponses [4,82-84]. They ac i a e ansc ip ion o cy okinin p ima y e- sponse genes; among hem he ype-A RRs. The exp es- siono ype-BRRs hemsel esisno induced h ough cy okinin signaling; hei ac i i y is egula ed h ough phospho yla ion o a conse ed Asp esidue in he e- cei e domain. A leas one A abidopsis B- ype RR, ARR2, is apidly deg aded upon i s cy okinin induced phopho y- la ion [85]. This mechanism p esumably p o ides p o e- olysis media ed eedback egula ion o i s ac i i y. In A abidopsis, he ype-B RRs sha e pa ially edundan unc ions; highe o de null mu an s show a p og essi ely dec eased sensi i i y o cy okinin [84,86,87]. The e a e six ype-B RR genes in P unus genome, whe eas Populus has hi een (P RR13-25) and A abi- dopsis ha e bo h wel e [88] (Figu e 6, Addi ional ile 9: Figu e S6). Two o he A abidopsis (ARR18 and ARR23) genes howe e code o a unca ed o m o he ecei e domain, hus hei unc ionali y as RRs is ques ionable. The s uc u e o Populus RR amily has p e iously been epo ed by Ramí ez-Ca ajal [20] and Pils and Heyl [14]. In hese wo epo s, al oge he 13 ype-B Populus RRs (P RR12-23) we e iden i ied, om which P RR12 is missing om he cu en assembly, whe eas P RR24 ep- esen s a newly iden i ied gene. Figu e 5 Un oo ed maximum likelihood ee o Populus (P ), P unus (Pp) and A abidopsis his idine phospho ans e (HP s) genes. The ee is based on a deduced amino acid (159 aa) sequence alignmen (Addi ional ile 8: Figu e S5). Suppo o each clade is gi en as ≥50% o boo s ap pseudo eplica es. Immanen e al. BMC Genomics 2013, 14:885 Page 7 o 12 h p://www.biomedcen al.com/1471-2164/14/885 Type-A RRs The p omo e s o ype-A RR genes con ain a high numbe o B- ype RR binding si es [82,83,89,90]. Acco dingly, phospho yla ed ype-B RRs ac i a e he exp ession o ype-A RR genes, which unc ion as nega i e eedback eg- ula o s o cy okinin signaling [8,91-93]. Type-A RRs may ep ess he ype-B RR ac i i y ei he by compe ing wi h hem o phospho ans e om ups eam HP s o by o ming inac i e he e odime s wi h hem [94]. Simila o he B- ype, in gene al, indi idual A- ype RRs a e also e- dundan in A abidopsis; sensi i i y o cy okinin inc eases p og essi ely in highe o de mu an s [86,87,91]). Ne e - heless, he e a e also pheno ypic di e ences be ween A a- bidopsis mu an s lacking mul iple ype-A RRs in di e en combina ions. A leas wo RRs o en sha e highly edun- dan unc ions, bu hese unc ions di e somewha om hose sha ed be ween he o he gene pai s [8,91-93]. I seems likely ha some unc ional speci ici y has e ol ed be ween he di e en RR genes and con ibu es o hei e en ion in plan genomes. The Populus genome con ains ele en ypes-A RRs (P RR1-11), whe eas P unus has only ou , compa ed o he engenesp esen in heA abidopsis(Figu e6, Addi ional ile 9: Figu e S6). Bo h Ramí ez-Ca ajal e al. [20] and Pils and Heyl [14] iden i ied hese same ype-A Populus RRs (Figu e 6). I appea s ha P unus has los membe s o his sub amily du ing i s e olu ion. Howe e , based on he ela i ely high edundancy be- ween A abidopsis RRs, his gene amily appea s o be well bu e ed agains loss o indi idual genes. Type-C RRs Type-C, o ex a, RRs ep esen a esponse egula o sub amily cha ac e ized by an a ypical ecei e domain amino acid sequence [80]. A abidopsis has wo o hem, ARR22 and ARR24. They display e y es ic ed exp es- sion pa e ns: ARR22 is exp essed exclusi ely in de elop- ing seeds [95], and ARR24 only in de eloping and ma u e pollen g ains [96]. ARR22 can in e ac and de- phospho yla e HP p o eins in i o; i hus appea s o ep esen a nega i e egula o o he cy okinin signaling phospho elay [78,95]. In con as o he ype-A RRs, he exp ession o ype-C RRs is no cy okinin inducible [78,96]. Ec opic o e -exp ession o ARR22 gi es ise o a dis inc pheno ype; he plan s a e dwa and s e ile [78]. Figu e 6 Un oo ed maximum likelihood ee o Populus (P ), P unus (Pp) and A abidopsis esponse egula o s (RRs). Membe s o ype-A, -B and –C RRs a e in ol ed in cy okinin signaling. Fo A abidopsis, also he pseudo esponse egula o genes wi h no known ole in cy okinin signaling a e included. The ee is based on a deduced amino acid (226 aa) sequence alignmen o ecei e (all RRs) and DNA-binding (GARP) (B- ype RRs) domains (Addi ional ile 9: Figu e S6). Suppo o each clade is gi en as ≥50% o boo s ap pseudo eplica es. Immanen e al. BMC Genomics 2013, 14:885 Page 8 o 12 h p://www.biomedcen al.com/1471-2164/14/885 Ye su p isingly, nei he a ege a i e no a ep oduc i e pheno ype was de ec ed in ei he single o double null mu an s o hese wo genes [95,96]. The unc ion o ype-C RRs emains elusi e. Compa ed o he wo A abidopsis genes, Populus has eigh ype-C RRs (P ARR26-33), whe eas in e es ingly, none was ound in he P unus genome (Figu e 6, Addi ional ile 9: Figu e S6). Pils and Heyl [14] iden i ied 10 unnamed ype- CPopulus RRs, ou o which ha e been emo ed om he new assembly; whe eas P RR28 and P RR33 ep esen newly iden i ied genes on ou lis . The expansion o ype- C sub amily appea s o be speci ic o he e olu ion o Populus lineage, as we know ha se e al o he species ha e less o hem: ice has only wo, and bo h maize and soybean ha e h ee [65]. All eigh Populus ype-C RRs ap- pea o sha e a common ances o wi h he wo A abidopsis ex a RRs. Two o hem, P RR27 and P RR29, ha e an a yp- ical conse ed mo i (HD-D-K and DD-E-K, espec i ely), and may ep esen pseudo esponse egula o s. Conclusions We epo he e he i s comp ehensi e desc ip ion o cy okinin signaling and homeos asis gene amilies in wo ha dwood ee species; Populus ichoca pa and P unus pe sica. Genomes o bo h species con ain he same cy o- kinin signal ansduc ion componen s as A abidopsis, e lec ing he ancien o igin o his ho mone signaling sys em. In gene al, he iden i ied gene amilies we e la - ge in Populus and smalle in P unus when compa ed o A abidopsis. In con as o he consensus a ios, some cy okinin signaling and homeos asis gene amilies ha e dis inc - i ely expanded in one o wo o he ee species as com- pa ed o A abidopsis. One o he expanded clades is he CKI1-like sub amily o wo-componen his idine kinases. This amily has h ee membe s in bo h Populus and P u- nus, as compa ed o a single gene in A abidopsis. This sha ed expansion indica es ha he gene numbe has p obably been mul iplied in a common ances o o he wo ee species. In e es ingly, in A abidopsis CKI1 has been shown o pa icipa e in he egula ion o bo h e- p oduc i e and seconda y ascula de elopmen . Fu u e esea ch will show i he ex a CKI1 o hologs ha e any ole in he con ol o cambial ac i i y and wood p oduc- ion in ee species. Ano he di e ence is seen in he HP gene amily, whe e bo h ee species ha e ou homologs o he single A abidopsis AHP4 gene. Ye , in his case, se e al o he species also ha e mul iple AHP4 homologs p esen in hei genomes. Some o hese homologs appea o ep e- sen pseudo HP s, which po en ially ac o inhibi he cy okinin phospho elay. I seems ha ha he e has been no ee lineage speci ic expansion, bu ha A abidopsis has ins ead los all bu one o i s AHP4 homologs. In con as o he changes sha ed by bo h ee species, some gene expansions appea o ha e aken place only in he Populus lineage. One clade o he LOG gene am- ily, he Populus o hologs o A abidopsis A LOG5,has expanded ou - old as compa ed o ei he o he wo o he species. Ano he gene sub amily, he C- ype RRs, has mul iplied ou - old in he Populus lineage as com- pa ed o A abidopsis, bu has ins ead disappea ed om P unus. Possibly o he RRs ha e eplaced unc ion o his RR class in P unus. As he unc ion o C- ype RRs has emained elusi e in A abidopsis, Populus could po- en ially u n ou o be a be e model o s udying hei ac i i y. We hope ha he iden i ica ion o cy okinin signaling and homeos asis pa hway om wo ha dwood ee spe- cies may se e as a e e ence upon which unc ional ana- lyses can be de eloped o de e mine he ole ha cy okinin plays in ege a i e and ep oduc i e ee de el- opmen . Addi ionally, hese genes may se e as po en ial candida e genes o ma ke -assis ed b eeding owa ds in- c eased wood and ui p oduc ion. Addi ional iles Addi ional ile 1: Table S1. Gene ic loci and gene models ( ansc ip IDs) o Populus ichoca pa cy okinin signaling and homeos asis genes based on he genome elease e sion 3.0 (h p://www.phy ozome.ne / sea ch.php?o g=O g_P ichoca pa_ 3.0). To enable compa isons wi h p e iously published Populus gene epo s, we ha e addi ionally included he espec i e loci and gene models as hey we e gi en in he assembly e sion 1.1. Addi ional ile 2: Table S2. Gene models and EST suppo o he P unus pe sica cy okinin signaling and homeos asis genes. The gene models a e gi en as in he genome elease e sion 1 (h p://www. phy ozome.ne /sea ch.php?me hod=O g_Ppe sica). Addi ional ile 3: Table S3. Lis o A abidopsis genes used in he cons uc ion o he phylogene ic ees. Addi ional ile 4: Figu e S1. Alignmen o Populus ichoca pa (P ), P unus pe sica (Pp) and A abidopsis cy okinin oxidase/dehyd ogenases (CKXs). Addi ional ile 5: Figu e S2. Alignmen o Populus (P ), P unus (Pp) and A abidopsis isopen enyl ans e ases (IPTs). Addi ional ile 6: Figu e S3. Alignmen o Populus (P ), P unus (Pp) and A abidopsis LONELY GUY (LOG) p o eins). Addi ional ile 7: Figu e S4. Alignmen o Populus (P ), P unus (Pp) and A abidopsis CRE1- and CKI1-like wo-componen his idine kinase, oge he wi h A abidopsis e hylene ecep o s (ETR1, ETR2, ERS1, ERS2 and EIN4), phy och omes (PHYA-E), a pu a i e osmosenso (A HK1), and he his idine kinase CKI2/AHK5. Addi ional ile 8: Figu e S5. Alignmen o Populus (P ), P unus (Pp) and A abidopsis his idine phospho ans e p o eins (HP s). The consensus HQxKGSSxS mo i , con aining he phospho-accep ing his idine esidue (H), is ma ked abo e he alignmen . Al oge he ou Populus (P HP 6a, P HP6b, PHP4b, and P HP-like), and wo P unus (PpHP6 and PpHP-like) HP s lack he conse ed his idine esidue. Addi ional ile 9: Figu e S6. Alignmen o Populus (P ), P unus (Pp) and A abidopsis esponse egula o s (RRs). Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Immanen e al. BMC Genomics 2013, 14:885 Page 9 o 12 h p://www.biomedcen al.com/1471-2164/14/885