Plan s 2015, 4, 183-195; doi:10.3390/plan s4020183
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A icle
De e mining he Composi ion o Lignins in Di e en Tissues o
Sil e Bi ch
Ku V. Fage s ed 1,*, Pekka Sa anpää 2, Ta ja Tapanila 2, Juha Immanen 1,3,
Juan An onio Alonso Se a 1 and Kaisa Nieminen 2
1 Depa men o Biosciences, Di ision o Plan Biology, P.O. Box 65, Uni e si y o Helsinki,
FI-00014 Helsinki, Finland; E-Mails: [email p o ec ed] (J.I.);
[email p o ec ed] (J.A.A.S.)
2 Na u al Resou ces Ins i u e Finland, Jokiniemenkuja 1, Fi-01300 Van aa, Finland;
E-Mails: pekka.sa anpää@luke. i (P.S.); [email p o ec ed] (T.T.);
[email p o ec ed] (K.N.)
3 Ins i u e o Bio echnology, P.O. Box 65, Helsinki Uni e si y, FI-00014 Helsinki, Finland
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +358-2941-59438.
Academic Edi o : Philip Ha is
Recei ed: 5 Decembe 2014 / Accep ed: 23 Ma ch 2015 / Published: 9 Ap il 2015
Abs ac : Quan i a i e and quali a i e lignin analyses we e ca ied ou on ma e ial om he
unks o sil e bi ch (Be ula pendula Ro h) ees. Two ypes o ma e ial we e analyzed.
Fi s , whole bi ch unk pieces we e c yosec ioned in o co k cambium, non-conduc i e
phloem, he cambial zone (conduc i e phloem, cambium and di e en ia ing xylem),
ligni ied xylem and he p e ious yea ’s xylem; ma e ial ha would show di e ences in lignin
amoun and quali y. Second, clonal ma e ial om one na u al bi ch popula ion was analyzed
o show a ia ions be ween indi iduals and be ween he lignin analysis me hods. The
di e en issues showed ma ked di e ences in lignin amoun and he sy ingyl:guaiacyl (S/G)
a io. In he non-conduc i e phloem issue con aining scle eids, he S/G a io was e y low,
and ypical o phloem ibe s and in he newly- o med xylem, as well as in he p e ious yea ’s
xylem, he a io lay be ween i e and se en, ypical o b oadlea ee xylem. Clonal ma e ial
consis ing o 88 s ems was used o calcula e he S/G a ios om he hioacidolysis and CuO
me hods, which co ela ed posi i ely wi h an R2 alue o 0.43. Compa isons o he me hods
indica e clea ly ha he CuO me hod is a good al e na i e o s udy he monome ic
composi ion and S/G a io o wood lignins.
OPEN ACCESS
Plan s 2015, 4 184
Keywo ds: ace yl b omide; Be ula pendula; cup ic oxide; lignin analysis me hods; phloem;
hioacidolysis; xylem
1. In oduc ion
Lignin is an a oma ic, he e ogenic cell wall he e opolyme ha is essen ial o mechanical suppo ,
wa e anspo and disease esis ance in ees [1]. Lignin can be ully emo ed, bu only unde d as ic
chemical condi ions using ei he s ong acid (e.g., sul i e pulping p ocess) condi ions unde ele a ed
empe a u es and p essu es o wi h ionic sol en s. As he emo al o lignin is cos ly, he e has been
conside able in e es in educing lignin con en by gene ic enginee ing o ees as a means o imp o ing
he e iciency o he pulping p ocess, educing he use o chemicals and imp o ing p o i abili y [2].
Lignin ende s wood xylem ecalci an in he bio e ine y p ocesses when bioe hanol is being p oduced.
Lignin is also a aluable polyme and se es as a pla o m chemical o a ious compounds, eplacing
ossil aw ma e ials, such as ca bon ibe s, dispe san s, anillin and new ypes o plas ics [3]. Hence,
i is impo an o know he lignin composi ion o unde s and how i can be p ocessed u he in o
new p oduc s.
Deciduous wood lignin is composed o sy ingyl (S) and/o guaiacyl (G) uni s linked by a se ies o
e he and ca bon-ca bon bonds. The e he β-O-4 linkages a e bo h equen and labile, which makes hem
he a ge o he deligni ica ion p ocess. In con as , he ca bon-ca bon linkages a e esis an , especially
he biphenyl 5–5 bonds in ol ing he a oma ic C-5 posi ion, which is a ailable o in e uni linkages
only in G uni s. Thus, coni e wood lignin essen ially made o G uni s is less suscep ible o k a
deligni ica ion han deciduous wood lignin [4].
The mos commonly-used me hods o quan i a i e analysis o lignin a e he Klason and AcB
me hods. Klason is a g a ime ic me hod and only measu es insoluble ma e ial a e hyd olysis wi h
72% H2SO4. I is o en combined wi h spec opho ome ic de e mina ion o dissol ed lignin [5].
Klason is ega ded as being gene ally eliable o woody s em ma e ial. Recen ly, his p ocedu e has
been modi ied o allow applica ion o a sample size o 50 mg [6]. The accu acy o lignin de e mina ion
depends also on issue and cell wall ype [7]. Va ious non-lignin componen s, such as annins,
non-ex ac ed polysaccha ides and p o eins [7,8], can be p esen in he lignin esidue and, hus, limi he
applicabili y o he Klason me hod in issues o he han xylem. The ace yl b omide me hod has been
epo ed o be he simples and as es among he me hods e alua ed, p esen ing simila o bes eco e y
o lignin in all issues assessed [9]. I is based on he solubiliza ion o lignin and he de e mina ion o
abso bance alues a 280 nm. The ace yl b omide p o ocol is based on he o ma ion o ace yl de i a i es
in non-subs i u ed OH g oups and b omide eplacemen o he Cα-OH g oups o p oduce a comple e
solubiliza ion o he cell wall ma e ial unde acidic condi ions. Howe e , an o e es ima ion o he lignin
con en can occu due o he oxida i e deg ada ion o s uc u al polysaccha ides (e.g., xylans) du ing he
incuba ion o he cell wall wi h he acid solu ion [9,10]. One di icul y wi h he ace yl b omide me hod
is he need o a well-de ined lignin s anda d o calib a e he me hod [7].
Chemical deg ada ion me hods a e wo hwhile when composi ional in o ma ion on lignin is needed.
Thioacidolysis has been applied o a wide a ie y o ma e ials and isola ed lignins [11,12]. Thioacidolysis
Plan s 2015, 4 185
is an acid-ca alyzed me hod ha esul s in β-O-4 clea age. The de ec ion o C6C3 p oduc s
(p-hyd oxyphenyl (H), guaiacyl (G) and sy ingyl (S) monome s) gi es in o ma ion on he composi ion
o non-condensed alkyl a yl s uc u es, and he esul s can be used o calcula ing he S/G- a ios.
Alkaline cup ic (II) oxide oxida ion (CuO) is a echnique commonly used o analyze he composi ion
o lignins in complex sample ma ixes, such as soils and sedimen s [13,14]. Rela i ely mild oxida ion
wi h CuO induces clea age o β-O-4 e he bonds in lignin. The lignin mac omolecule is hyd olyzed,
yielding phenolic CuO oxida ion p oduc s wi h aldehydic, ke onic and acidic side chains [15–17].
The deg ada ion p oduc s ob ained by bo h hioacidolysis and CuO me hods can be analyzed by a a ie y
o means, including gas ch oma og aphy/mass spec ome y (GC/MS). Nei he hioacidolysis no he
CuO me hod comple ely depolyme ize lignin, and he e o e, he esul does no co e he o al con en
o lignin, bu gi es aluable in o ma ion abou he lignin subs uc u es.
The aim o he p esen piece o wo k is o s udy he composi ions o bi ch lignins using bo h he CuO
and hioacidolysis me hods. Fo his pu pose, we chose he wood ma e ial ca e ully: Fi s , o use he
me hods on wood ma e ial o na u al a iabili y, we used 22 di e en bi ch clones c ea ed o ep esen
di e en geno ypes ound in na u al s ands. Second, o es ima e how la ge a ia ions in he sample
anging om ou e phloem o ma u e xylem will a ec lignin composi ion, we used longi udinal c yosec ions
ca e ully cu om all o he di e en issues o he sil e bi ch unk. Hence, we had ma e ial ha
con ained di e en ypes o lignin (phloem ibe s s. seconda y xylem), high amoun s o pec in, p o eins
and hemicelluloses (cambial egion) and issues wi h high concen a ions o ha dwood- ype lignin
wi h a high S/G- a io. Ou majo aim was o es he eliabili y and pe inence o he CuO and
hioacidolysis me hods.
2. Resul s and Discussion
2.1. Lignin Composi ion o Di e en Sil e Bi ch Tissue F ac ions
Di e en issues we e c yosec ioned om 13-yea -old whole sil e bi ch s ems and di ided in o
i e ac ions (Figu e 1). The ac ions we e subjec ed o CuO ea men o clea e β-O-4 e he bonds
in lignin. The phenolic compounds de i ed a e shown in Figu es 2–4. The ou majo g oups include
guaiacyl (G), sy ingyl (S), hyd oxycinnamyl (C) and p-hyd oxyphenyl (H) moie ies.
The ac ions showed dis inc i e di e ences in he p oduc s o CuO ea men (Figu e 2; no e he
di e en scales in he g aphs). Aldehydes ( anillin and sy ingaldehyde) we e he majo p oduc s, wi h
mos o he anillin in he non-conduc i e phloem, while sy ingaldehyde was he majo p oduc in he
ligni ied xylem. The acid con en ( anillic and sy ingic acid) inc eased om co k cambium o he xylem.
La ge a ia ions be ween he samples we e no iceable in he acids.
4-Hyd oxy compounds we e he p oduc s de ec ed in F ac ions 1 o 3, bu he e we e only aces in
he xylem. Wi hin he hyd oxycinnamyl g oup, e ulic acid was he domina ing compound wi h mino
amoun s in he xylem (Figu e 3). The amoun o p-couma ic inc eased om he co k cambium o he
cambium and was de ec ed only in mino amoun s in he xylem. The amoun o e ulic acids in all
ac ions was a abou he same le el, excep in he cambial zone (Figu e 3). Fe ulic acid has been shown
o ake pa in he lignin polyme , and i has been pos ula ed as an ini ia ion o nuclea ion si e a he
beginning o lignin polyme iza ion [18].
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Figu e 1. Pho omic og aphs o he sil e bi ch sample sec ions wi h a c yomic o ome.
(A) A c oss-sec ion h ough he s em. Mainly cellulosic cell walls a e s ained blue wi h
Alcian blue and ligni ied issues ed wi h sa anin. (B) Longi udinal sec ions p esen ing he
ac ual samples o lignin analyses: 1, co k cambium; 2, non-conduc i e phloem; 3, combined
conduc i e phloem, cambium and di e en ia ing xylem; 4, ligni ied xylem; 5, p e ious
yea ’s xylem.
Figu e 2. Con .
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A anillin
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B ace o anillone
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C anillic acid
Plan s 2015, 4 187
Figu e 2. Guaiacyl (A–C) and sy ingyl (D–F) uni s eleased by he CuO ea men o
he di e en issues analyzed wi h he GC-MS. 1, Co k cambium; 2, non-conduc i e phloem;
3, combined conduc i e phloem, cambium and di e en ia ing xylem; 4, ligni ied xylem;
5, p e ious yea ’s xylem.
Figu e 3. p-Hyd oxyl phenols (A–C) and hyd oxycinnamyls (D,E) eleased by he CuO
ea men . 1, Co k cambium; 2, non-conduc i e phloem; 3, combined conduc i e phloem,
cambium and di e en ia ing xylem; 4, ligni ied xylem; 5, p e ious yea ’s xylem.
Figu es 4 and 5 show he amoun s pe g o d y weigh o he S-, G-, P- and C-g oups and he S/G
a ios in he di e en ac ions. The S-g oup con ains sy ingaldehyde, ace osy ingone and sy ingic acid,
he G-g oup anillin, ace o anillone and anillic acid, he P-g oup 4-hyd oxybenzaldehyde,
4-hyd oxyace ophenone and 4-hyd oxybenzoic acid and he C-g oup p-couma ic acid and e ulic acid.
The S/G a io was low in he co k cambium and non-conduc i e phloem, in e media e in he cambial
egion and high in he xylem. In he cambial egion, he S- and G-g oup compounds we e a a low le el.
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E ace osy ingone
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F sy ingic acid
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A 4-hyd oxybenzaldehyde
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B 4-hyd oxyace ophenone
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C 4-hyd oxybenzoic acid
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D p-couma ic acid
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E e ulic acid

Plan s 2015, 4 188
Figu e 4. To al amoun s pe g o d y weigh o he sy ingyl (S)-, he guaiacyl (G)-,
p-hyd oxyphenyls (H)- and hyd oxycinnamyls (C)-g oups in he di e en ac ions ob ained
using he CuO me hod. (A) The S-g oup con ains sy ingaldehyde, ace osy ingone and
sy ingic acid; (B) he G-g oup anillin, ace o anillone and anillic acid; (C) he P-g oup
4-hyd oxybenzaldehyde, 4-hyd oxyace ophenone and 4-hyd oxybenzoic acid; and (D) he
C-g oup p-couma ic acid and e ulic acid. 1, Co k cambium; 2, non-conduc i e phloem;
3, combined conduc i e phloem, cambium and di e en ia ing xylem; 4, ligni ied xylem;
5, p e ious yea ’s xylem.
Figu e 5. The S/G a ios as calcula ed om he CuO analysis esul s. 1, Co k cambium;
2, non-conduc i e phloem; 3, combined conduc i e phloem, cambium and di e en ia ing
xylem; 4, ligni ied xylem; 5, p e ious yea ’s xylem.
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S/G, CuO
Plan s 2015, 4 189
Figu e 6B shows ha he o al lignin con en s, de e mined using he ace yl b omide me hod, o he
di e en ac ions we e simila . This is in acco dance wi h he sums o he monome concen a ions
eleased by he CuO me hod (Figu e 6A) o all o he o he ac ions, excep F ac ion 3. This ac ion,
which con ained he cambial egion whe e ligni ica ion is likely o be a he minimum, yielded he lowes
sum o monome s. The absence o lignin in his issue can also be seen in he s aining in Figu e 1. This
egion con ains p edominan ly cellulose, hemicelluloses, pec in and p o eins [19]. This disc epancy
be ween Figu e 6A,B could be pa ially explained by he ac ha he p-hyd oxycinnamic acids a e
bi unc ional molecules wi h ca boxylic and phenolic binding si es, and hey can be in ol ed in bo h es e
and e he linkages o o he cell wall componen s. I is also ue ha deg aded xylans (due o pe chlo ic
acid) ha e s ong abso bance a 280 nm, leading o o e es ima ion o lignin wi h he AcB me hod. The
cambial egion is high in p o ein con en [19], bu p o eins p ecipi a e du ing sample p epa a ion and
should no in e e e wi h he AcB me hod.
Figu e 6. The same sil e bi ch issues analyzed by CuO and AcB me hods. (A) The sum
o he monome s eleased by he CuO me hod; (B) he o al lignin amoun es ima ed by he
AcB me hod. 1, Co k cambium; 2, non-conduc i e phloem; 3, combined conduc i e
phloem, cambium and di e en ia ing xylem; 4, ligni ied xylem; 5, p e ious yea ’s xylem.
2.2. Va ia ion in Lignins in a Na u al Sil e Bi ch Popula ion
Figu e 7 shows a compa ison o CuO and hioacidolysis in he es ima ion o he S/G a io. The
posi i e co ela ion was a he low wi h R2 being 0.43 and he linea eg ession equa ion
y = 0.681x + 0.6635. I was obse ed ha he S/G alues ob ained wi h he hioacidolysis me hod we e
in mos cases highe han hose calcula ed om CuO esul s (a e age CuO = ca. 80% o hioacidolysis).
The in e es ing and la ge a ia ion in he S/G a io in he sil e bi ch xylem ma e ial is explained by he
ac ha he ma e ial p esen ed he la ge na u al a ia ion wi hin a na u al bi ch popula ion. I would be
o in e es o see whe he his is e lec ed in he pulping p ope ies o he wood ma e ial. Howe e , he
o al lignin con en (Klason + acid-soluble lignin) showed e y li le a ia ion, and mos samples ell
om 23% o 25% o d y weigh . The sum o he analy ical monome s eleased by he CuO me hod
eached 25% o 55% o he Klason lignin. In ea lie accoun s, hioacidolysis co e s ca. 20%–40% o he
weigh o he lignin p esumably p esen [20].
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A CuO-lignin
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B AcB -lignin
Plan s 2015, 4 190
Figu e 7. Compa isons o he S/G a ios ob ained om he CuO (S/G, CuO) and
hioacidolysis (S/G, TiO) analysis esul s. The wood ma e ial o igina ed al oge he om
88 ees o 22 di e en geno ypes p esen ing na u al a ia ion in sil e bi ch.
3. Expe imen al Sec ion
Fo he di e en issues o he sil e bi ch unk (Be ula pendula Ro h), we elled h ee bi ch ees a
he expe imen al ield o he Viikki Campus a he Uni e si y o Helsinki. The ees we e o he clone
No. V5834 c ea ed om ma e ial o he Punkaha ju expe imen al si e o he Finnish Fo es Resea ch
Ins i u e. The ees we e 13 yea s old, ca. 10 m in heigh and hei diame e ca. 11 cm a a 1.5-m heigh .
Longi udinal sec ions (20 μm in hickness) we e cu in he angen ial di ec ion in whole bi ch unk
pieces wi h a c yomic o ome and di ided in o i e ac ions—1, co k cambium; 2, non-conduc i e
phloem; 3, conduc i e phloem, cambium and di e en ia ing xylem; 4, ligni ied xylem; 5, p e ious yea ’s
xylem— o show di e ences in lignin amoun s and quali y be ween he issues. Fo mic oscopy, he
sec ions we e s ained wi h 1% sa anin and hen 1% Alcian blue, which s ain ligni ied cell walls ed and
unligni ied cell walls blue, espec i ely.
A ield expe imen in Punkaha ju, sou heas e n Finland (61°48' N, 29°18' E), was es ablished in 1999
o long- e m moni o ing o wi hin-s and di e ences be ween sil e bi ch (Be ula pendula Ro h)
geno ypes in g ow h phenomena. The expe imen was plan ed on abandoned o me ag icul u al land
wi h a ine sandy ill soil and had a andomized comple e block design wi h 6 eplica e blocks. All blocks
con ained ou eplica e ees o he 22 geno ypes a a plan ing dis ance o 2 × 2 m. The geno ypes we e
mic o-p opaga ed om ees selec ed andomly in one s and (one hec a e in size) o B. pendula ha was
na u ally egene a ed a e logging ope a ions in 1979. In 2008, one ee pe each geno ype in ou
andomly selec ed blocks was elled, al oge he 88 ees. Sample discs we e cu a b eas heigh (1.3 m).
Th ee– ou o he ou e mos annual ings we e emo ed om wo ca dinal di ec ions (sou h and no h)
in o de o a oid any ungal in ec ion and decay nea he pi h. The samples we e u he cu in o
2 × 2-mm s icks.
y = 0.681x + 0.6635
R² = 0.4276
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S/G, CuO
S/G, TiO
Plan s 2015, 4 191
3.1. Quan i a i e Analysis o Lignin
3.1.1. Klason and Acid-Soluble Lignin De e mina ion
Samples we e g ound ozen wi h a blade-mill (Polymix PX-A10). The d y solids con en o he
milled wood samples was de e mined a 103 °C. The samples o ai -d ied wood powde s (3 g) we e
ex ac ed wi h ace one using a Soxhle appa a us o 6 h [21]. A e e apo a ion o he sol en s, he
esidues we e d ied a 103 °C, allowed o cool in a desicca o and hen weighed. The amoun o
acid-insoluble lignin was de e mined by he Klason me hod [5]. The samples o he ex ac ed wood
powde s (300 mg) we e ea ed wi h 3 cm3 o 72% sul u ic acid unde acuum o 1 h. The mix u es
we e dilu ed wi h abou 82-cm3 po ions o wa e and au ocla ed a 125 °C o 1 h. The p ecipi a es
we e collec ed wi h glass ibe il e SS GF 52 × 47 mm by suc ion il a ion and washed wi h wa e .
The il e s wi h he acid-insoluble lignin (Klason lignin) we e d ied a 103 °C, cooled in he desicca o
and weighed. In o de o de e mine he amoun o acid-soluble lignin, he il a es we e dilu ed wi h
wa e o 250 cm3. Abso p ion o he acid solu ions wi h he dissol ed lignin was measu ed a 203 nm
using sul u ic acid o he same concen a ion as a blank.
The abso bance eadings we e ob ained wi h a Shimadzu UV-2401 PC UV-VIS Reco ding
spec opho ome e . The o al lignin (Klason lignin + acid-soluble lignin) con en was calcula ed om
he unex ac ed wood as ollows: Klason lignin% = p(100 − u)/m, in which p = p ecipi a e (g),
u = ex ac i es (%) and m = calcula ed d y weigh o ex ac ed sample (g). The acid-soluble lignin
con en was calcula ed using a lignin abso p i i y o 128 L·g−1·cm−1 and co ec ed because o he
abso p ion o ca bohyd a es acco ding o [21]. The o al lignin con en o each sample was de e mined
as he mean o he duplica e measu emen s; his is e e ed o as he measu ed lignin con en o
he sample.
3.1.2. AcB -Based De e mina ion
The powde was ei he lyophilized (bi ch issues) o d ied a 60 °C o 48 h (bi ch clones), and 5- o
10-mg samples we e ex ac ed wi h ace one o emo e soluble ex ac i es. Lignin con en s we e
de e mined using he ace yl b omide me hod [22,23]. A sample o 5 o 10 mg o phloem and inne ba k
issue was weighed in o a 10-mL sc ew-capped es ube and sonica ed wi h 5 mL o ace one o 30 min.
The ex ac was pipe ed o , and he ex ac i e ee sample was d ied. I was edissol ed in 5 mL
20% ( / ) AcB -ace ic acid solu ion (con aining 100 µL 70% pe chlo ic acid, a haza dous chemical) and
he sample was kep in a block hea e a 50 °C o 3 h wi h egula shaking [22]. A e ea men , he
sample was ozen a −20 °C o 15 min in o de o s op he eac ion. The mel solu ion was ans e ed
o a 50-mL olume ic lask con aining 5 mL 2 M NaOH and 12 mL 100% ace ic acid. The solu ion was
dilu ed in o 50 mL wi h ace ic acid. The UV spec um was measu ed wi h a Shimadzu UV-2401
spec ome e a 280 nm. Lignin con en was calcula ed using he ollowing exp ession:
Lignin% = 100(As − Ab)V/aW
As = abso bance o sample
Ab = abso bance o blank
V = olume o solu ion