BioMed Cen al
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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.
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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
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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.
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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.
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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.

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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.
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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.
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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.
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(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.