Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birch

BioMed Cen al Page 1 o 15 (page numbe no o ci a ion pu poses) BMC Plan Biology Open Access Resea ch a icle Phenolic compounds in ec omyco hizal in e ac ion o lignin modi ied sil e bi ch Su i Su ela*1, Ka oliina Niemi2, Jaanika Edesi1, Tapio Laakso3, Pekka Sa anpää3, Jaana Vuosku1, Riina Mäkelä1, Heidi Tiimonen4, Vincen L Chiang5, Janne Koskimäki1, Ma ja Suo sa1, Rii a Julkunen-Tii o6 and Hely Häggman1 Add ess: 1Depa men o Biology, Uni e si y o Oulu, PO Box 3000, 90014 Oulu, Finland, 2Depa men o Applied Biology, Uni e si y o Helsinki, PO Box 27, 00014 Helsinki, Finland, 3Finnish Fo es Resea ch Ins i u e, Van aa Resea ch Uni , Jokiniemenkuja 1, 01301 Van aa, Finland, 4Finnish Fo es Resea ch Ins i u e, Punkaha ju Resea ch Uni , Finlandian ie 18, 58450 Punkaha ju, Finland, 5Fo es Bio echnology Resea ch G oup, Depa men o Fo es y and En i onmen al Resou ces, College o Na u al Resou ces, No h Ca olina S a e Uni e si y, Campus Box 7247, 2500, Pa ne s II Building, Raleigh, NC 27695-7247, USA and 6Depa men o Biology, Uni e si y o Joensuu, PO Box 111, 80101 Joensuu, Finland Email: Su i Su ela* - su i.su ela@oulu. i; Ka oliina Niemi - ka oliina.nie[email p o ec ed]; Jaanika Edesi - [email protected]; Tapio Laakso - apio.laakso@me la. i; Pekka Sa anpää - pekka.sa anpaa@me la. i; Jaana Vuosku - jaan[email p o ec ed]; Riina Mäkelä - iina.mak[email p o ec ed]; Heidi Tiimonen - heidi. ii[email p o ec ed]; Vincen L Chiang - [email p o ec ed]; Janne Koskimäki - janne.koskimaki@oulu. i; Ma ja Suo sa - ma ja_suo sa@ho mail.com; Rii a Julkunen-Tii o - ii a.julkunen- [email p o ec ed]; Hely Häggman - hely.haggm[email p o ec ed] * Co esponding au ho Abs ac Backg ound: The monolignol biosyn he ic pa hway in e connec s wi h he biosyn hesis o o he seconda y phenolic me aboli es, such as cinnamic acid de i a i es, la onoids and condensed annins. The objec i e o his s udy is o e alua e whe he gene ic modi ica ion o he monolignol pa hway in sil e bi ch (Be ula pendula Ro h.) would al e he me abolism o hese phenolic compounds and how such al e a ions, i exis , would a ec he ec omyco hizal symbiosis. Resul s: Sil e bi ch lines exp essing quaking aspen (Populus emuloides L.) ca ea e/5- hyd oxy e ula e O-me hyl ans e ase (P COMT) unde he 35S cauli lowe mosaic i us (CaMV) p omo e showed a educ ion in he ela i e exp ession o a pu a i e sil e bi ch COMT (BpCOMT) gene and, consequen ly, a dec ease in he lignin sy ingyl/guaiacyl composi ion a io. Al e a ions we e also de ec ed in concen a ions o ce ain phenolic compounds. All P COMT sil e bi ch lines p oduced no mal ec omyco hizas wi h he ec omyco hizal ungus Paxillus in olu us (Ba sch: F .), and he o ma ion o symbiosis enhanced he g ow h o he ansgenic plan s. Conclusion: The down- egula ion o BpCOMT in he 35S-P COMT lines caused a educ ion in he sy ingyl/guaiacyl a io o lignin, bu no signi ican e ec was seen in he composi ion o quan i y o phenolic compounds ha would ha e been caused by he exp ession o P COMT unde he 35S o UbB1 p omo e . Mo eo e , he de ec ed al e a ions in he composi ion o lignin and seconda y phenolic compounds had no e ec on he in e ac ion be ween sil e bi ch and P. in olu us. Published: 29 Sep embe 2009 BMC Plan Biology 2009, 9:124 doi:10.1186/1471-2229-9-124 Recei ed: 20 Feb ua y 2009 Accep ed: 29 Sep embe 2009 This a icle is a ailable om: h p://www.biomedcen al.com/1471-2229/9/124 © 2009 Su ela 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. BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 2 o 15 (page numbe no o ci a ion pu poses) Backg ound The phenylp opanoid pa hway gi es ise o a a ie y o compounds ha a e used in he biosyn hesis o cinnamic acid de i a i es, lignin, la onoids and condensed an- nins. These phenolic compounds o m a di e se g oup o seconda y me aboli es, exhibi ing nume ous biological unc ions in plan s. In he Be ula species, he phenolic compound concen a ions a y acco ding o he de elop- men phase o he plan [1,2], clone [2-4] o plan pa [5] and o di e en en i onmen al ac o s [2,4,5]. Mo eo e , annins and speci ic la onoids ha e been shown o play a ole in de ence agains he bi o y [6] and p o ec ion agains UVB adia ion [1,7-9]. In addi ion o he phenolic compound p o iles o di e en Be ula species, he gene al ou line o he phenylp opanoid pa hway o he species is also well known [4]. The seconda y cell wall is essen ial o he conduc ion o wa e and he s uc u al in eg i y o ascula plan s as well as o de ence agains insec he bi o es and pa hogens. The seconda y cell wall is composed o mul iple laye s o cellulose mic o ib ils embedded in a ma ix o hemicellu- lose, lignin and pec in. Lignin, p obably he mos s udied phenolic compound, is composed o monome s de i ed om he monolignol biosyn he ic pa hway [10]. In ha d- woods, coni e yl, sinapyl and p-couma yl alcohol a e he main lignin monome s, gi ing ise o guaiacyl (G), sy ingyl (S) and p-hyd oxyphenyl (H) phenylp opanoid uni s, espec i ely, when polyme ized o he lignin mole- cule. These hyd oxycinnamyl alcohols di e in hei deg ee o me hyla ion and, consequen ly, o m a ying linkage ypes in he lignin, de e mining he solubili y o he polyme . In sinapyl alcohol, he C-5 posi ion o he a oma ic ing is me hyla ed, which p e en s he o ma ion o s ong linkage ypes ha a e ypical o G uni s. Angiospe m lignin consis s mainly o G and S monome s and is mo e easily deligni ied han he G uni ich gymno- spe m lignin. The monolignol biosyn he ic pa hway is s ill unde deba e, pa ly because he enzymes in ol ed in he pa hway a e mul i unc ional and exhibi b oad sub- s a e speci ici y, a leas in i o, making se e al al e na i e eac ion o de s possible. The mos upda ed iew o he angiospe m monolignol biosyn he ic pa hway is p e- sen ed by Li e al. [11], Do e al. [12] and Vanholme e al. [13]. The ca ea e/5-hyd oxy e ula e O-me hyl ans e ase (COMT) (EC 2.1.1.68), also known as 5-hyd oxyconi e yl aldehyde O-me hyl ans e ase (AldOMT) [14] ca alyses he me hyla ion o he C-5 posi ion o angiospe ms' S p e- cu so s. COMT belongs o he plan Class II O-me hyl- ans e ases (OMTs) oge he wi h enzymes ha me hyla e nume ous phenolic compounds, such as phe- nylp openes and la onols [15,16]. Ini ially COMT was shown o use ca eic acid and 5-hyd oxy e ula e as sub- s a es [17,18], bu u he s udies demons a ed ha COMT is especially in ol ed in he biosyn hesis o S lignin [19-22] and, u he mo e, ha he me hyla ion occu s a 5-hyd oxyconi e aldehyde and (o ) 5-hyd oxy- coni e yl alcohol as shown wi h a ious angiospe m spe- cies [14,23-26]. Howe e , he subs a e p e e ences o COMT may a y be ween species being, o ins ance, ela- i ely boa d in al al a (Medicago sa i a L.) [24] and whea (T i icum aes i um L.) [26]. Some o he enzymes ha ing COMT ac i i y a e p obably bi unc ional as in he case o A abidopsis haliana OMT (A 5g54160) which is in ol ed in bo h lignin and la onoid biosyn hesis [12,27,28]. Sil e bi ch (Be ula pendula Ro h) is one o he key species in bo eal o es ecosys ems and, in addi ion, economically he mos impo an deciduous ee species in No dic coun ies. In Finland, based on he na ional o es in en- o y pe o med du ing yea s 2004 h ough 2007 app oxi- ma ely 16% o g owing s ock was bi ch (363 mill. m3) [29]. The bi ch oundwood is used as a aw ma e ial in he chemical pulp indus y bu also in plywood p oduc ion. Mo eo e , bi ch is an impo an sou ce o ene gywood: in 2007 wood-based uels co e ed one i h (295PJ) o he o al ene gy consump ion in Finland [30]. Bo eal o es ees li e in a mu ualis ic associa ion wi h ec omyco hizal (ECM) ungi, which enables g ow h in he nu ien -poo , acidic soils. The o ma ion o ECM symbiosis causes changes in he ansc ip ion le els o bo h pa ne s [31-34], esul ing in mo phological and physiological al e a ions. The p oli e a ion o oo hai s is inhibi ed and he epide mal cells o eede oo s in angiospe ms elonga e adially as he ungus pene a es in o he in e cellula space o he epide mis. The ungal hyphae ha co e eede oo s a e also a sou ce o an ex e nal hyphal ne . These dis inc i e al e a ions in he symbio ic pa ne s ensu e he e ec i e exchange o wa e and nu ien s om he ungal pa ne o he ca bohy- d a es o he plan [35]. ECM o ma ion has also been obse ed o al e he exp ession le els o genes in ol ed in he phenylp opanoid pa hway [32,33,36] and he con- cen a ions o phenolic compounds [37-43]. Howe e , he esul s ha e been a he inconsis en . T ansgenic plan s ha e g ea po en ial o u u e ag icul- u e, sil icul u e and bio uel p oduc ion. Inc easing he pes and disease esis ance o plan s as well as imp o ing wood quali y and enhancing wood p oduc ion ha e been he a ge s o bo h con en ional b eeding and gene ic enginee ing. F om an indus ial poin o iew, lignin qual- i y and con en a e o pa icula in e es . The emo al o lignin in chemical pulping is a cos ly p ocess which could be acili a ed wi h mo e soluble lignin and lowe lignin con en [44]. A educ ion in lignin con en would also be bene icial o he p oduc ion o bioe hanol [45]. O he BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 3 o 15 (page numbe no o ci a ion pu poses) p ocesses ela ed o he p oduc ion o bioe hanol could also be enhanced by modi ica ions in he cell wall chem- is y, as e iewed by S icklen [46]. Lignin modi ica ions using a ious gene cons uc s ha a e associa ed wi h he monolignol biosyn he ic ou e ha e been conduc ed suc- cess ully on angiospe m ee species ( e iewed in [10,11,13]). Changes in he p ima y as well as in he seconda y me ab- olism o o ganisms a e igge ed by a a ie y o s imuli, such as changes in he de elopmen al phase o en i on- men al ac o s. The e o e, he pleio opic o non- a ge e ec s o ansgenes should also be s udied in di e se en i onmen al condi ions. So a , only mino changes ha e been ound in in e ac ions be ween lignin modi ied ees and he bi o es o soil mic o auna [47-53]. Recen s udies in es iga ing possible changes in he seconda y me abolism ha a e caused by gene ic ans o ma ions ha e mos ly been conduc ed on he baceous species [54- 59] and wi hou he in ol emen o ecological in e ac- ions. In he p esen s udy, we analyzed he phenolic com- pounds and lignin cha ac e is ics o P COMT sil e bi ch lines (Be ula pendula Ro h.) in in e ac ion wi h he ECM ungus Paxillus in olu us (Ba sch: F .) in o de o de e mine he impac o he symbiosis on he phenylp opanoid ou e de i ed compounds and o de ec possible unin- ended e ec s o ansgene exp ession. Resul s Exp ession o P COMT and BpCOMT in oo s The open eading ame o pu a i e COMT (BpCOMT) and pa ial sequence (1536 bp) o PP2A (BpPP2A) o sil- e bi ch we e sequenced. The pu a i e BpCOMT was 72% iden ical o P COMT [EMBL: X62096] a he nucleo ide le el and 87% iden ical a he amino acid le el (Addi- ional ile 1) and showed highes simila i y o he cas o bean (Ricinus communis) COMT [GenBank: EEF36570] (90%) and almond (P unus dulcis) COMT [EMBL: CAA58218] (88%). The pu a i e BpPP2A showed 91% simila i y wi h Medicago sa i a subsp. x a ia [GenBank: AAG29593] and 90% simila i y wi h A. haliana [Gen- Bank: NP_172790] PP2A a he amino acid le el (Addi- ional ile 2). The exp ession o P COMT and he pu a i e BpCOMT was s udied om he non-inocula ed and myc- RT-PCR esul s o BpCOMT and P COMT in sil e bi ch oo sFigu e 1 RT-PCR esul s o BpCOMT and P COMT in sil e bi ch oo s. Rela i e exp ession o he endogenous pu a i e ca ea e/5-hyd oxy e ula e O-me hyl ans e ase o sil e bi ch (BpCOMT) (A) and he he e ologous P COMT gene (B) no mal- ized using a ub and pu a i e BpPP2A as e e ence genes in he non-inocula ed and myco hizal oo s o clone A and P COMT- modi ied lines 23, 44 and 65. Values a e means ± s anda d e o . Di e en le e s abo e he columns deno e signi ican (P < 0.05) di e ence be ween he P COMT lines and clone A wi hin he ea men s acco ding o he wo-sample - es o he Wil- coxon ank sum es wi h he Bon e oni co ec ion. Numbe o eplica es 3-5. BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 4 o 15 (page numbe no o ci a ion pu poses) o hizal oo s o sil e bi ch (Figu e 1A, B). The ela i e exp ession o he pu a i e BpCOMT was simila in bo h non-inocula ed and myco hizal oo s: 35S-P COMT lines 23 and 44 had lowe a e age le els o BpCOMT ansc ip s han UbB1-P COMT line 65 and clone A (Figu e 1A). Howe e , signi ican di e ences (P < 0.05) in he ela i e exp ession o BpCOMT we e only obse ed in he non- inocula ed oo s be ween UbB1-P COMT line 65 and 35S- P COMT line 23. The ela i e exp ession o P COMT was signi ican ly (P < 0.05) highe in he non-inocula ed oo s o 35S-P COMT line 23 han in line 65, whe e he ans- gene was d i en by he UbB1 p omo e (Figu e 1B). In myco hizal oo s, he ela i e exp ession le els o P COMT be ween lines we e compa able o hose o non- inocula ed oo s. Lignin dis ibu ion and composi ion Lignin con en as a pe cen age o d y weigh (DW) e alu- a ed wi h he ace yl b omide me hod was 27.6% in he non-inocula ed and 27.1% in he myco hizal oo s o clone A. In P COMT lines he oo lignin con en a ied be ween he highes alue o 27.8% o myco hizal oo s o line 65 and he lowes o 24.5% o myco hizal oo s o line 23. The co esponding lignin con en s o s em wood we e mo e han 5 pe cen age uni s lowe han he oo lignin con en s and a ied be ween 19.5 and 23.5%. Nei- he he ansgene no he ungal ea men a ec ed he lignin con en . The GC-MS analyses o lignin uni s showed ha he non-inocula ed clone A had highe (P < 0.05) S/G a ios in bo h s em and oo wood han he non-inocula ed plan s o P COMT line 44 (Figu e 2A and 2B). In non-inocula ed oo s o P COMT line 23 he S/G a io was lowe (P < 0.05) han in he oo s o clone A. The S/G a io o s em and oo wood o myco hizal P COMT line 44 was signi ican ly educed (P < 0.05) in compa ison wi h he myco hizal clone A. In he s em and oo wood o bo h non-inocula ed and myco hizal P COMT line 65, he S/G a ios we e a he same le el as in clone A. Mo e- o e , in he s em and oo wood o non-inocula ed P COMT line 65, he S/G a ios we e signi ican ly (P < 0.05) highe han in he co esponding non-inocula ed P COMT lines 23 and 44. Acco ding o he Mäule assay he S lignin (i.e. he pink- ed colou a ion) was only sligh ly educed when he oo and s em xylem sec ions o P COMT lines 23 (Figu e 3F, N) and 44 (Figu e 3G, O) The lignin sy ingyl/quiaicyl a ios o non-inocula ed and myco hizal sil e bi chesFigu e 2 The lignin sy ingyl/quiaicyl a ios o non-inocula ed and myco hizal sil e bi ches. The lignin sy ingyl/quiaicyl (S/G) a ios o s ems (A) and oo s (B) o non- ansgenic clone A and P COMT-modi ied lines 23, 44 and 65. Values a e means ± s anda d de ia ion. Di e en le e s abo e he columns deno e signi ican (P < 0.05) di e ences be ween he non-inocula ed and myco hizal plan s wi hin he line/clone and be ween lines/clone wi hin he ungal ea men acco ding o he Wilcoxon ank sum es wi h he Benjamini & Hochbe g co ec ion o he wo-sample - es wi h he Benjamini & Hochbe g co ec ion. Numbe o eplica es 3. BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 5 o 15 (page numbe no o ci a ion pu poses) we e compa ed o he xylem sec ions o clone A (3E, M) and P COMT line 65 (3H, P). Soluble phenolic compounds and condensed annins No clone- o line-speci ic peaks we e de ec ed in he HPLC-DAD o HPLC-MS ch oma og ams and, mo eo e , all phenolic compounds we e p esen in he non-inocu- la ed and myco hizal samples o clone A and P COMT lines (Table 1 and Addi ional ile 3). Ace yla ed kaemph- e ol, my ice in and que ce in wi h hamnoside moie y we e ound in all lea es o clone A and P COMT lines. Condensed annin concen a ions we e high in he sam- ples (Table 1). The annin le els pa ly p e en ed he iden- i ica ion o soluble phenolic componen s, especially om he oo samples (Addi ional ile 3). In he lea es o myco hizal plan s, signi ican di e ences (P < 0.05) we e ound in he concen a ions o que ce in 3-a abinose and kaemphe ol 3-ace yl-glucoside be ween P COMT lines 44 and 65 (Addi ional ile 3). Signi ican di e ences be ween clone A and he P COMT lines we e ound in he concen a ion o p-OH-cinnamic acid de i- a es, indi idual cinnamic acid de i a i es 3 and 4 and chlo ogenic acid and chlo ogenic acid de i a i e. A signi - ican di e ence (P < 0.05) was de ec ed in he amoun o (+)-ca echin in he lea es o he myco hizal and non- inocula ed plan s o clone A. In s ems, he cinnamic acid de i a i es we e a a highe (P < 0.05) le el in clone A han in P COMT lines 23 and 44 (Table 1). O indi idual componen s, he concen a ion His ochemical localiza ion o lignin in non-inocula ed sil e bi chesFigu e 3 His ochemical localiza ion o lignin in non-inocula ed sil e bi ches. C oss-sec ions o s em and oo o non-inocu- la ed clone A (A, E, I, M) and P COMT-modi ied lines 23 (B, F, J, N), 44 (C, G, K, O) and 65 (D, H, L, P). S ems (A-H) and oo s (I-P). Lignin s ained pink- ed in he phlo oglucinol-HCL s ained sec ions (A-D, I-L). In he Mäule s ainings (E-H, M-P), sy ingyl lignin pink- ed and guaiacyl lignin ligh b own o da k b own. x , xylem ib e; x , xylem ay; x , xylem essel. BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 6 o 15 (page numbe no o ci a ion pu poses) o p-OH-cinnamic acid glucoside was highe (P < 0.05) in he s ems o he clone A han in he s ems o P COMT lines 23 and 44 (Addi ional ile 3). Signi ican di e ences we e also de ec ed be ween he P COMT lines and clone A in he concen a ions o galloca echin, 3,4'-dihyd oxyp o- piophenone 3-glucoside (DHPPG) and (+)-ca echin, which was a a highe le el in he s ems o clone A han in he s ems o P COMT lines. The concen a ion o condensed annin p ecu so s was signi ican ly (P < 0.05) highe in he myco hizal oo s o line 23 han in he oo s o lines 44 and 65 (Table 1). A Table 1: Concen a ions o phenolic compounds and condensed annins in non-inocula ed and myco hizal sil e bi ches Lea es Clone Lines T A 23 44 65 Cinnamic acid de i a i es c 2.48 ± 0.44 a 2.12 ± 0.57 a 2.21 ± 0.37 a 2.30 ± 0.43 a ECM 2.84 ± 0.71 a 2.35 ± 0.72 a 2.35 ± 0.41 a 2.50 ± 0.54 a Fla onoids c 26.14 ± 4.00 a 30.67 ± 8.64 a 28.85 ± 2.14 a 30.87 ± 8.68 a ECM 27.07 ± 7.36 a 25.38 ± 9.78 a 25.80 ± 5.25 a 29.71 ± 5.80 a Apigenin de i a i es c 0.20 ± 0.12 a 0.31 ± 0.10 a 0.31 ± 0.09 a 0.43 ± 0.25 a ECM 0.40 ± 0.21 a 0.19 ± 0.11 a 0.24 ± 0.07 a 0.39 ± 0.12 a Kaemphe ol de i a i es c 0.85 ± 0.13 a 0.71 ± 0.23 a 0.90 ± 0.19 a 0.68 ± 0.20 a ECM 0.83 ± 0.14 a 0.73 ± 0.22 a 0.97 ± 0.13 a 0.69 ± 0.06 a My ice in de i a i es c 18.31 ± 3.58 a 22.81 ± 6.34 a 21.77 ± 1.75 a 21.66 ± 6.11 a ECM 18.83 ± 5.04 a 18.40 ± 8.20 a 17.90 ± 4.30 a 20.17 ± 4.54 a Que ce in de i a i es c 5.19 ± 1.34 a 6.34 ± 2.12 a 5.43 ± 1.45 a 7.05 ± 1.64 a ECM 6.38 ± 1.52 a 5.52 ± 1.98 a 5.84 ± 1.18 a 7.20 ± 1.19 a Condensed annins c 160.41 ± 24.97 a 142.87 ± 47.63 a 127.54 ± 34.23 a 154.82 ± 28.44 a ECM 142.40 ± 68.98 a 106.7 ± 22.57 a 149.21 ± 24.76 a 142.32 ± 62.23 a p-OH-cinnamic acid de i a i es c 3.21 ± 0.47 a 1.84 ± 0.49 b 2.46 ± 0,32 ab 2.71 ± 0.61 ab ECM 3.10 ± 0.36 ac 2.22 ± 0.71 bc 2.42 ± 0.32 ac 2.42 ± 0.38 ab S ems Clone Lines T A 23 44 65 Cinnamic acid de i a i es c 2.68 ± 0.68 a 1.37 ± 0.28 b 1.33 ± 0.25 b 1.65 ± 0.37 bc ECM 2.33 ± 0.33 ac 1.46 ± 0.18 b 1.27 ± 0.30 b 1.18 ± 0.20 b Fla onoids c 14.33 ± 1.74 ab 11.43 ± 1.49 ab 10.23 ± 1.43 b 13.79 ± 2.81 ab ECM 14.60 ± 2.12 a 12.38 ± 1.89 ab 10.77 ± 2.37 ab 11.18 ± 1.47 ab Phenolic glycosides c 16.86 ± 1.46 ab 19.48 ± 0.81 ab 18.18 ± 2.22 ab 22.67 ± 5.31 b ECM 15.40 ± 2.74 a 20.61 ± 2.29 ab 20.61 ± 2.29 ab 17.37 ± 5.29 ab Condensed annins c 108.57 ± 50.76 a 138.57 ± 13.52 a 137.43 ± 13.72 a 141.47 ± 16.70 a ECM 144.82 ± 19.68 a 132.86 ± 11.23 a 131.90 ± 9.26 a 130.32 ± 12.55 a Roo s Clone Lines T A 23 44 65 Cinnamic acid de i a i es c 0.71 ± 0.33 a 0.17 ± 00.05 a 0.17 ± 0.10 a 0.37 ± 0.23 a ECM 0.53 ± 0.30 a 0.19 ± 0.02 a 0.20 ± 0.13 a 0.24 ± 0.14 a Fla onoids c 11.39 ± 0.62 a 9.45 ± 1.83 a 8.41 ± 1.93 a 8.26 ± 2.26 a ECM 11.24 ± 2.78 a 12.10 ± 2.55 a 7.88 ± 2.91 a 7.21 ± 1.25 a Gallo/Ellagi annins c 0.06 ± 0.01 a 0.32 ± 0.24 a 0.28 ± 0.09 a 0.12 ± 0.06 a ECM 0.11 ± 0.06 a 0.36 ± 0.08 a 0.23 ± 0.12 a 0.15 ± 0.13 a Condensed annins c 130.50 ± 22.43 a 113.87 ± 17.86 a 106.92 ± 27.75 a 112.24 ± 8.94 a ECM 126.09 ± 6.80 a 116.24 ± 10.11 a 108.47 ± 5.62 a 100.46 ± 2.24 a Condensed annin p ecu so s c 35.07 ± 5.81 ab 31.44 ± 7.15 ab 28.60 ± 5.50 ab 28.50 ± 3.81 ab ECM 37.20 ± 9.43 ac 41.92 ± 10.62 a 20.38 ± 6.70 b 25.44 ± 2.21 bc Concen a ions (mg/DW g) o phenolic compounds and condensed annins in he lea , s em and oo samples o sil e bi ch clone A and P COMT- modi ied lines 23, 44 and 65 a e 8 weeks in co-cul u e wi h P. in olu us. Values a e means ± s anda d de ia ions in he p esence (ECM) o absence (c) o he ungus. Di e en le e s ollowing he alues deno e signi ican di e ences (P < 0.05) be ween he non-inocula ed and myco hizal plan s wi hin he line/clone and be ween lines/clone wi hin he ungal ea men acco ding o he K uskal-Wallis es combined wi h he Wilcoxon ank sum es wi h he Benjamini & Hochbe g co ec ion o he one-way o wo-way Ano a combined wi h Tukey's hones ly signi ican di e ence es o wi h he wo-sample - es wi h he Benjamini & Hochbe g co ec ion. Fo s a is ical es ing he lea apigenin de i a i es we e squa e oo , s em cinnamic acid de i a i es log 10 and oo condensed annins squa e ans o med. Numbe o eplica es 4-7. BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 7 o 15 (page numbe no o ci a ion pu poses) small amoun o ellagic acid was ound in P COMT lines 23 and 44, whe e he P COMT was d i en by he 35S p o- mo e , bu no in lines 65 and clone A (Addi ional ile 3). An ellagic acid de i a i e was also ound in line 65 and in he myco hizal oo s o clone A. Fo ma ion o ECMs and g ow h cha ac e is ics o sil e bi ches All P COMT lines we e able o o m ECM symbiosis wi h P. in olu us, and inocula ion esul ed in sligh ly highe su i al pe cen ages in clone A and P COMT lines 23 and 44 (Table 2). The myco hizal pe cen ages o inocula ed plan s a ied conside ably be ween P COMT lines and clone A. No di e ences we e de ec ed in he numbe o ECMs pe oo sys ems (Table 2) o in he mo phology o he myco hizas be ween lines: well-de eloped hyphal man le co e ed he oo ips and he epide mal cells we e adially elonga ed and su ounded by ungal Ha ig ne (Figu e 4A-D). Compa ed wi h he esh weigh s (FWs) o he P COMT-modi ied lines, plan s o clone A had lowe FWs, bu he g ow h a e (i.e. inal FW/ini ial FW a io) o he clone A plan s in bo h ea men s co esponded o he g ow h o he ansgenic lines (Table 3). Inocula ion o P COMT-modi ied lines wi h P. in olu us enhanced hei g ow h, esul ing in signi ican ly (P < 0.05) highe FWs han ha o he non-inocula ed plan s (Table 3). The oo /shoo a ios o plan s inc eased signi ican ly (P < 0.05) as a esul o inocula ion in clone A and P COMT line 23. Inocula ion had no e ec on he numbe o leng h o ad en i ious oo s. Discussion In he p esen s udy, no changes we e ound in phenolic compounds o P COMT-modi ied sil e bi ch lines ha would ha e been caused by he o ma ion o ECM symbi- osis wi h P. in olu us. The only di e ence be ween he myco hizal and non-inocula ed plan s was obse ed in he ca echin concen a ion in he lea es o he non- ans- genic clone A. Münzenbe ge e al. [39,40] obse ed a educ ion in a ious phenolic compounds (e.g. p- hyd oxybenzoic acid glucoside, picein and ca echin) in he myco hizal ine oo s o Eu opean la ch (La ix decidua Mill.) and No way sp uce [Picea abies (L.) Ka s .] when compa ed wi h he non-myco hizal oo s. Simila esul s we e ob ained wi h Eu opean beech (Fagus syl a ica L.) ECM oo s which con ained less ca echin [41]. By con- as , an inc ease in ca echin concen a ion was de ec ed in he ECM oo s o Eu opean la ch [42] and he needles and s ems o myco hizal Sco s pine (Pinus syl es is L.) [43]. Fu he mo e, enhanced le els o phenolic com- pounds ha e been obse ed in Douglas- i [Pseudo suga menziesii (Mi b.) F anco] [60] and b own ba el (Eucalyp- us as iga a Deane and Maiden) [37]. The disc epancy o esul s may e lec he luc ua ion o ansc ip ome pa - e ns du ing ECM o ma ion, as seen in a ious mic oa - ay expe imen s [32-34], di e se biological ma e ial and expe imen al designs. In he p esen wo k, all P COMT lines we e able o o m symbiosis wi h mo phologically no mal ECMs. Mo eo e , he myco hizal in e ac ion inc eased FWs in all P COMT lines. Simila esul s ha e been ob ained wi h sil e bi ches exp essing suga bee chi inase IV [61] and 4-couma a e: coenzyme A ligase (4CL) [52] and P COMT [53]. In all o hese s udies, ans- genic sil e bi ches we e capable o o ming ECM symbi- osis al hough 4CL exp essing sil e bi ches had changes in hei g ow h cha ac e is ics [52] and wo P COMT sil e bi ch lines had al e ed ECM mo phology in i o [53]. In sil e bi ch-P. in olu us in e ac ion, Feugey e al. [62] obse ed a ansien inc ease in phenylalanine ammonia- lyase (PAL) ac i i y, bu in he mic o-a ay s udies [31,33] PAL was no di e en ially exp essed in ECM oo s compa ed wi h non-inocula ed oo s. Ins ead, Le Qué é e al. [33] ound an inc ease in genes coding monolignol biosyn hesis ou e associa ed p oduc s: A abidopsis ca e- oyl-coA 3-O-me hyl ans e ase (CCoAOMT) homolog, di igen p o ein homolog and sinapyl alcohol dehyd oge- nase (SAD) homolog. CCoAOMT exp ession was consis - en a e 4 days o inocula ion o 14 days, whe eas he exp ession o di igen p o ein homolog and SAD homolog was a i s highes a e 2 days o inocula ion and hen again 14 days a e he s a o he co-cul i a ion. Ou esul s indica e ha ECM o ma ion had no d as ic e ec Table 2: Su i al and ECM cha ac e is ics o sil e bi ches ECM ca ego ies Clone/ Line Su i al % o non-inocula ed plan s Su i al % o inocula ed plan s ECM % I II III IV V A74 763132121 23 92 95 83 11 8 3 4 3 44 95 100 74 14 2 6 3 3 65 92 87 58 8 5 3 1 2 Su i al pe cen ages o non-inocula ed and inocula ed sil e bi ches o clone A and P COMT-modi ied lines 23, 44 and 65, pe cen ages o he ECM plan s o all inocula ed plan s (ECM %) and numbe o ECM oo ips in oo sys ems classi ied o i e ca ego ies: I = 1-20 ECMs, II = 20-30 ECMs, III = 30-50 ECMs, IV = 50-100 ECMs, V ≥ 100 ECMs. BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 8 o 15 (page numbe no o ci a ion pu poses) on he lignin o he phenolic compound biosyn hesis in s ems o oo s. The exp ession o P COMT unde con ol o he 35S p o- mo e esul ed in lowe S/G a ios in he s em and oo wood when compa ed wi h clone A, as obse ed in p e i- ous s udies [48,53,63]. By con as , when P COMT was unde he UbB1 p omo e , no changes we e de ec ed in he lignin cha ac e is ics. We ha e p e iously shown [48,63] ha he e a e mul iple copies o he P COMT gene in lines 23, 44 and 65 and ha he UbB1-P COMT- an- sc ip is bigge han he 35S-P COMT- ansc ip . In he p esen s udy, he ela i e exp ession o he he e ologous P COMT seemed o be highe in he oo s o 35S-P COMT lines 23 and 44 han in hose o UbB1-P COMT line 65. Con e sely, he BpCOMT mRNA ansc ip le els we e mo e dec eased in 35S-P COMT lines 23 and 44 han in UbB1-P COMT-line 65. The homology be ween BpCOMT and P COMT a he nucleo ide le el was qui e high and i is he e o e possible ha he he e ologous P COMT exp ession esul ed in RNAi-media ed pa ial silencing o he endogenous BpCOMT. The ela i e exp ession le els o BpCOMT and P COMT possibly indica e ha he 35S p omo e gene a ed a highe numbe o mRNA ansc ip s o P COMT han UbB1 and, as a consequence, dec eased he numbe o BpCOMT ansc ip s mo e in ensi ely in 35S-P COMT lines 23 and 44, hus causing a educ ion in he lignin S/G a io. The monolignol biosyn he ic pa hway c oss alks wi h o he cell wall associa ed pa hways [64,65] and also wi h he biosyn he ic pa hways o a ious phenolic com- pounds [66-70] which sha e he same p ecu so s. Conse- quen ly, he al e ed exp ession o monolignol biosyn he ic pa hway genes may esul in changes in he lignin con en and phenolic compound p o iles as shown wi h supp essed COMT and CCoAOMT (EC 2.1.1.104) [66], cinnamoyl-CoA educ ase (CCR; EC 1.2.1.44) [68], hyd oxycinnamoyl-CoA shikima e/quina e hyd oxycin- namoyl ans e ase (HCT; EC 2.3.1.133) [69] and cinna- ma e 4-hyd oxylase (C4H; 1.14.13.11) [67,70]. In he p esen s udy, chemical changes we e de ec ed be ween he P COMT lines and he non- ansgenic clone A in he concen a ions o phenolic compounds in he oo s, s ems and lea es o bo h non-inocula ed and myco hizal plan s. The de ec ed changes we e p obably no di ec esul s o he ansgene because 35S-P COMT lines 23 and 44 displayed di e ences in phenolic compound p o iles. Fu he mo e, he changes in he phenolic p o iles o lea es, s ems and oo s a e wi hin he na u al a ia ion o phenolic compounds wi hin sil e bi ch [1-3,5,7]. Conclusion In he p esen s udy, he down- egula ion o BpCOMT in he 35S-P COMT lines caused no shi o monolignol pa hway in e media es o he biosyn hesis o he phenolic seconda y compounds. Mo eo e , no appa en e ec in he composi ion o quan i y o phenolic compounds caused by he exp ession o P COMT unde he 35S o UbB1 p omo e could be ound. To conclude, ou esul s indica e ha he p esen lignin modi ica ion in he P COMT lines does no a ec phenolic p o iles o he symbio ic ela ionship be ween sil e bi ch and P. in olu- us. Me hods Plan and ungal ma e ial Sil e bi ch (Be ula pendula Ro h.) lines 23, 44 and 65 exp essing he ca ea e/5-hyd oxy e ula e O-me hyl ans- e ase (P COMT) gene [EMBL: X62096] o quaking aspen Table 3: G ow h cha ac e is ics o non-inocula ed and myco hizal sil e bi ches Clone/ Line T Ini ial FW (g) Final FW (g) Ra io o inal and ini ial FW Roo /shoo FW a io Numbe o ad en i ous oo s Leng h o ad en i ous oo s (cm) A c 0.08 ± 0.04 a 1.75 ± 0.76 a 25.79 ± 19.90 a 0.80 ± 0.25 a 4.00 ± 1.33 a 16.70 ± 2.83 a ECM 0.07 ± 0.02 a 2.03 ± 0.76 a 34.49 ± 17.44 a 0.97 ± 0.24 b 4.00 ± 1.31 a 17.14 ± 1.04 a 23 c 0.15 ± 0.08 a 2.76 ± 0.88 a 23.75 ± 16.23 a 1.39 ± 0.40 a 4.97 ± 1.49 a 17.55 ± 3.78 a ECM 0.16 ± 0.09 a 3.06 ± 0.86 b 23.06 ± 13.63 a 1.59 ± 0.48 b 5.50 ± 1.72 a 17.59 ± 3.35 a 44 c 0.14 ± 0.06 a 2.70 ± 0.37 a 22.93 ± 9.28 a 1.40 ± 0.26 a 5.15 ± 1.54 a 18.64 ± 2.00 a ECM 0.15 ± 0.08 a 2.89 ± 0.51 b 23.28 ± 12.30 a 1.43 ± 0.29 a 7.68 ± 3.16 a 19.07 ± 2.10 a 65 c 0.17 ± 0.08 a 2.59 ± 0.43 a 17.70 ± 7.03 a 1.19 ± 0.20 a 4.03 ± 0.85 a 19.52 ± 2.05 a ECM 0.18 ± 0.09 a 2.83 ± 0.47 b 20.73 ± 11.90 b 1.26 ± 0.31 a 4.53 ± 1.22 a 18.52 ± 1.85 a E ec s o he myco hiza o ma ion on he g ow h o sil e bi ch con ol clone A and P COMT-modi ied lines 23, 44 and 65 a e 8 weeks in co- cul u e wi h P. in olu us in a g eenhouse. Ini ial esh weigh s (FWs) and inal FWs o plan s, oo /shoo a ios, numbe and leng h o ad en i ious oo s. Values a e means ± s anda d de ia ions in he p esence (ECM) o absence (c) o he ungus. Di e en le e s ollowing he alues deno e a signi ican di e ence (P < 0.05) be ween he non-inocula ed and inocula ed plan s wi h myco hizas wi hin each line/clone acco ding o he Wilcoxon ank sum es o he wo-sample - es . Numbe o eplica es 9-35. BMC Plan Biology 2009, 9:124 h p://www.biomedcen al.com/1471-2229/9/124 Page 9 o 15 (page numbe no o ci a ion pu poses) (Populus emuloides L.) [17] we e gene a ed as desc ibed by A onen e al. [63] and Tiimonen e al. [48]. The P COMT encodes Class II me hyl ans e ase (EC 2.1.1.68), which uses 5-hyd oxyconi e yl aldehyde as a p ima y subs a e [14]. All ansgenic lines we e p oduced om clone A as desc ibed in Valjakka e al. [71], o igina - ing in Punkaha ju, Eas e n Finland (61°48' N, 29°17' E). In P COMT lines 23 and 44, he ansgene was d i en by he 35S cauli lowe mosaic i us (CaMV) p omo e and in line 65 by he sun lowe polyubiqui in (UbB1) p omo e . The gene cons uc s we e pRT99/35S-P COMT and pRT99/UbB1-P COMT, espec i ely. The plan s o all lines ECM oo s o sil e bi chFigu e 4 ECM oo s o sil e bi ch. C oss-sec ions (5-10 μm) o sil e bi ch clone A (A) and P COMT lines 23 (B), 44 (C) and 65 (D) oo s a e 8 weeks o co-cul i a ion wi h P. in olu us. A ow, Ha ig ne ; iangle, mycelium o P. in olu us; e, epide mal cell o sil e bi ch oo . Ba s = 20 μm.