Mul imodal imaging analysis in sil e i e eals
coo dina ion in cellulose and lignin deposi ion
Gonzalo Pé ez-de-Lis ,
1,2,
* Béa ice Richa d ,
2
Fabienne Quilès ,
3
Au élie De eau ,
4
Igna ius-K is ia Adiku nia ,
2
Cy ille B.K. Ra hgebe
2
1 BIOAPLIC, Depa amen o de Bo ánica, Uni e sidade de San iago de Compos ela, EPSE, Campus Te a, 27002 Lugo, Spain
2 Uni e si é de Lo aine, Ag oPa isTech, INRAE, SILVA, F-54000 Nancy, F ance
3 Uni e si é de Lo aine, CNRS, LCPME, F-54000 Nancy, F ance
4 Uni e si é de Lo aine, INRAE, IAM, F-54000 Nancy, F ance
*Au ho o co espondence: [email p o ec ed]
The au ho esponsible o dis ibu ion o ma e ials in eg al o he indings p esen ed in his a icle in acco dance wi h he policy desc ibed in he
Ins uc ions o Au ho s (h ps://academic.oup.com/plphys/pages/Gene al-Ins uc ions) is Gonzalo Pé ez-de-Lis ([email p o ec ed]).
Abs ac
Despi e lignin being a key componen o wood, he dynamics o acheid ligni ica ion a e gene ally o e looked in xylogenesis
s udies, which hampe s ou unde s anding o en i onmen al d i e s and blu s he in e p e a ion o iso opic and ana omical
signals s o ed in ee ings. He e, we analyzed cell wall o ma ion in sil e i (Abies alba Mill.) acheids o de e mine i cell wall
ligni ica ion lags behind seconda y wall deposi ion. Fo his pu pose, we applied a mul imodal imaging app oach combining
ansmi ed ligh mic oscopy (TLM), con ocal lase scanning mic oscopy (CLSM), and con ocal Raman mic ospec oscopy
(RMS) on ana omical sec ions o wood mic oco es collec ed in no heas F ance on 11 da es du ing he 2010 g owing season.
Wood au o luo escence a e lase exci a ion a 405 and 488 nm associa ed wi h he RMS sca e ing o lignin and cellulose,
espec i ely, which allowed iden i ica ion o ligni ying cells (cells showing ligni ied and nonligni ied wall ac ions a he
same ime) in CLSM images. The numbe o ligni ying cells in CLSM images mi o ed he numbe o wall- hickening bi e ingen
cells in pola ized TLM images, e ealing highly synch onized kine ics o wall hickening and ligni ica ion (simila imings and
du a ions a he cell le el). CLSM images and RMS chemical maps e ealed a subs an ial inco po a ion o lignin in o he wall a
ea ly s ages o seconda y wall deposi ion. Ou esul s show ha mos o he cellulose and lignin con ained in he cell wall unde -
go concu en pe iods o deposi ion. This sugges s a s ong synch oniza ion be ween cellulose and lignin- ela ed ea u es in
coni e ee- ing eco ds, as hey o igina ed o e highly o e lapped ime ames.
Open Access
Recei ed No embe 29, 2023. Accep ed Ma ch 18, 2024. Ad ance access publica ion Ap il 9, 2024.
© The Au ho (s) 2024. Published by Ox o d Uni e si y P ess on behal o Ame ican Socie y o Plan Biologis s.
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 ps://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed euse, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Resea ch A icle
In oduc ion
Lignin is he second mos abundan g oup o na u al polyme s
on Ea h a e cellulose and ep esen s up o 30% o he
o ganic ca bon in he biosphe e (Boe jan e al. 2003). This im-
plies ha a subs an ial pa o he ca bon aken by he ee
lea es is seques e ed in he o m o lignin. I also plays a majo
ole in he physiology o ascula plan s, con e ing comp es-
si e s eng h and wa e impe meabili y o suppo ing and
conduc ing xylem cells, which accoun s o hei supe io
mechanical and hyd aulic esis ance (Vanholme e al. 2010;
Voelke e al. 2011; Pe ei a e al. 2018). Lignin also con ibu es
o woody plan s’ de ense agains pa hogens, inc easing hei
esis ance o deg ada ion by mic obial a ack (Ranade e al.
2022). On he o he hand, mul iple indus ial uses o lignocel-
lulosic ma e ials highligh he majo economic impo ance o
lignin biosyn hesis, including he de elopmen o ma e ials
such as wood–polyme composi es, lignin-based adhesi es,
nanopa icles, o ca bon ibe s among o he s (No g en and
Edlund 2014; Leng e al. 2022). Howe e , al hough ee- ing
esea ch has con ibu ed o shedding ligh on he
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en i onmen al cons ain s o ligni ica ion (Pie ma ei e al.
2020), he unde pinnings o he in e ac ion be ween xylem
de elopmen al ac o s and clima e a e s ill a om being
unde s ood.
Lignin is a phenolic he e opolyme syn hesized du ing xylo-
genesis, which is cha ac e ized by he ollowing phases: cell di -
ision, cell enla gemen , wall hickening (i.e. seconda y cell wall
[SCW] deposi ion and cell wall ligni ica ion), and p og ammed
cell dea h (Ra hgebe e al. 2016). A e di ision, mo he xylem
cells expe ience i e e sible lumen enla gemen h ough p i-
ma y cell wall (PCW) ex ension, a e which he S1, S2, and S3
laye s o he SCW and he wa y laye a e o med.
Ligni ica ion begins in ini ia ion si es loca ed in cell co ne
(CC) and middle lamella (in e ace be ween 2 adjacen ac-
heids), sp eading la e inwa d in o he SCW (Donaldson 2001;
Schmi e al. 2003) and concluding a e cell dea h (Pesque
e al. 2013; Meen s e al. 2018). Du ing ligni ica ion, in e lamella
oids le by cellulose mic o ib ils a e illed by lignin, which
o ms chemical bonds wi h hemicelluloses. The ein, mono-
lignols p e iously syn he ized in he cell lumen a e inco po a ed
in o polyme s h ough he ac ion o pe oxidases (Boe jan e al.
2003; Vanholme e al. 2010; Tobima su e al. 2013). SCW depos-
i ion is known o occu a e cell enla gemen , esul ing in a lag
e ec be ween g ow h in size and biomass in ee s ems
(And ianan enaina e al. 2019). Likewise, p e ious s udies con-
e ge on he idea ha ligni ica ion ollows SCW deposi ion.
Howe e , he p ecise ime lag be ween hese 2 phases a he
cell and issue le els emains la gely unknown. Some au ho s a -
gued ha he SCW would be la gely o e en o ally de eloped
be o e ligni ica ion (Donaldson 1991; Fukushima and
Te ashima 1991; Donaldson 2001), while mo e ecen s udies
sugges ha ligni ica ion may ini ia e soon a e he polysac-
cha ide ma ix s a s o be deposi ed (Joseleau and Ruel 2006;
Meen s e al. 2018). This knowledge gap hampe s an accu a e
conside a ion o he con ibu ion o cellulose and lignin o
he in aannual dynamics o s em biomass g ow h.
Examina ion o wood mic oco es epea edly collec ed
h oughou he g owing season by using ansmi ed ligh mi-
c oscopy (TLM) has con ibu ed o a mechanis ic unde s and-
ing o xylogenesis p ocesses and hei espec i e en i onmen al
d i e s (Cuny e al. 2019). In gymnospe ms, he assessmen o
in aannual dynamics o wood o ma ion has been possible
hanks o he applica ion o a da a-d i en modeling app oach
on di e en ia ing and ma u e cell coun s ob ained along adial
iles om ans e se mic osec ions (Cuny e al. 2013). Changes
in he adial numbe o acheids ac oss ma u e, wall- hickening,
enla ging, and cambial zones o e ime can be used o es ima e
he ime spen by acheids in consecu i e di e en ia ion
phases (Fig. 1). Howe e , cells unde going SCW deposi ion
and ligni ica ion ha e o en been included wi hin he same wall-
hickening zone, using (i) pola ized ligh o lag he onse o
SCW deposi ion hanks o bi e ingence o cellulose mic o ib ils
and (ii) di e en dye combina ions o ma k he end o ligni ica-
ion (Ra hgebe e al. 2016). As a esul , in aannual dynamics o
SCW deposi ion and ligni ica ion ha e a ely been compa ed,
obscu ing he in e p e a ion o in e - and in a ing se ies o
ela ed ai s (Pé ez-de-Lis e al. 2022). This could be in pa a -
ibu ed o di icul y in de ec ing ea ly ligni ica ion s ages due o
limi ed con as and esolu ion achie ed in TLM images (Bond
e al. 2008), which ad oca es o he use o mo e powe ul im-
aging ools o s udy ligni ica ion.
Ad anced imaging echniques (e.g. ansmission elec on mi-
c oscopy) ha e been used o ende a p ecise e alua ion o SCW
deposi ion and ligni ica ion in bo h coni e s (Schmi e al. 2003;
G iča e al. 2005) and angiospe ms (P islan e al. 2009). Xylem
composi ion has also been analyzed by le e aging on na u al
wood luo escence (Donaldson 2013, 2020), employing echni-
ques like ul a iole mic ospec opho ome y (e.g. Schmi
e al. 2003). Con ocal lase scanning mic oscopy (CLSM) is an-
o he luo escence-based ool used o assess cell wall ul as uc-
u e (G ünwald e al. 2002; Donaldson e al. 2010; Ki in e al.
2020), as well as o moni o ligni ica ion (Dickson e al. 2017;
Nanayakka a e al. 2019; Balzano e al. 2021). This has been pos-
sible because lignin polyphenolic a oma ic ings p oduce luo -
escen signals ollowing exci a ion by ul a iole and isible
ligh s. Ye , wood au o luo escence does no only depend on lig-
nin, as cellulose and hemicellulose unsa u a ed glycosidic bonds
also emi luo escence (Ding e al. 2020). Thus, some au ho s
ha e p oposed o label lignin wi h luo escen dyes and an i-
bodies o isualize lignin- ich issues (Ki in e al. 2003; Joseleau
and Ruel 2006; Bond e al. 2008; Tobima su e al. 2013; Ki in
e al. 2020; Balzano e al. 2021). On he o he hand, analy ical
ools such as con ocal Raman mic ospec oscopy (RMS) ha e
been used o s udy he chemis y o cell wall, as i ende s spa ial
in o ma ion on he dis ibu ion o lignin and o he cell wall con-
s i uen s (Aga wal 1999; Gie linge e al. 2012; Guillon e al. 2022;
Leng e al. 2022). The e o e, combining TLM-de i ed obse a-
ions wi h spa ial in o ma ion p o ided by CLSM and RMS could
b ing aluable in o ma ion on he dynamics o ligni ica ion.
This s udy aimed o analyze SCW deposi ion and ligni ica-
ion in sil e i (Abies alba Mill.) acheids h ough a mul i-
modal imaging app oach. Fi s , we analyzed he p esence o
lignin on ans e se wood mic osec ions by using RMS and
CLSM images. To assess he o se be ween wall hickening
(i.e. SCW deposi ion and cell wall ligni ica ion) and ligni ica-
ion, we cha ac e ized hei espec i e dynamics (Fig. 1)
h ough adial cell coun s ob ained om TLM and CLSM
images. Th ee main hypo heses we e es ed: (i) a he issue
le el, lignin concen a ions dec ease om ma u e xylem o-
wa d he cambial zone acco ding o he deg ee o cell di e -
en ia ion; (ii) a he cell le el, SCW deposi ion s a s ea lie
han cell wall ligni ica ion; and (iii) imings and du a ions o
wall hickening and ligni ica ion espec i ely ob ained
h ough TLM and CLSM e lec he delay be ween SCW de-
posi ion and cell wall ligni ica ion.
Resul s
Cha ac e is ic Raman and au o luo escence signals in
ma u e xylem and he ascula cambium
The RMS spec um eco ded a he compound middle
lamella (CML) o a ma u e acheid (Fig. 2) showed he spec al
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ea u es o he 3 s uc u al polyme s o xylem (cellulose,
hemicelluloses, and lignin). I is also wo h no ing he con i-
bu ion o he spec um o His olaque p o eins h ough he
Phe b ea hing mode o he a oma ic ing a 1,003 cm
−1
.
The lignin band a 1,599 cm
−1
was he mos p ominen o
he spec um (Fig. 2), exhibi ing an in eg a ed in ensi y
5- old highe han he one o cellulose. Mo eo e , he
RMS chemical image showed ha lignin was well dis ibu ed
ac oss he ma u e acheid wall (Fig. 2), wi h celluloses, hemi-
celluloses, and backg ound sca e ing ela ed o His olaque
p o eins consis en ly showing a mo e limi ed in ensi y
(Supplemen a y Fig. S1). Con e sely, he RMS spec um ob-
ained om he hin angen ial wall o a cambial cell lacked
he spec oscopic signa u e o lignin a 1,599 cm
−1
(Fig. 3),
whe eas he o he wood componen s (i.e. cellulose and
hemicelluloses) we e de ec ed conjoin ly o His olaque
p o eins.
The au o luo escence emi ed by he xylem was mainly
due o cell walls, ollowed by he p o oplas o li ing cells
(Fig. 4). A s ong luo escen emission occu ed in ma u e
acheids a 442 o 513 nm a e exci a ion wi h
he 405 nm lase beam ( ep esen ed in blue in composi e
CLSM images, Fig. 4, Supplemen a y Fig. S2), while ha in
cambial cells was e y weak and lagged up o 504 o
575 nm. This emission was p esen h ough he en i e cell
wall, allowing easy ecogni ion o ea lywood and la ewood
acheids (Supplemen a y Fig. S2). A weak luo escen emis-
sion signal was cap u ed in bo h ma u e acheids (513 o
575 nm) and cambial and phloem cells (531 o 600 nm) o
he 488 nm lase beam ( ep esen ed in yellow in CLSM
images, Fig. 4, Supplemen a y Fig. S2). Thus, while mos o
he ma u e xylem au o luo escence emission was exci ed a
405 nm, he au o luo escence o phloem and cambial cell
walls was emi ed only a e exci a ion by he 488 nm lase
beam. Mic osec ions occasionally showed auma ic esin
duc s and epi helial cells, which luo escence emission oc-
cu ed exclusi ely upon exci a ion a 488 nm (Fig. 4).
His olaque p o eins did no exhibi luo escence emission
a e exci a ion by 405 o 488 nm lase beams.
Cell-le el changes in Raman and au o luo escence
signals du ing xylem di e en ia ion
In de eloping xylem, RMS hype spec al maps we e eco ded in
he CC and CML o e 9 di e en loca ions wi hin he wall-
hickening (1 o 7) and enla ging (8 o 9) zones (Fig. 5). The
RMS spec a om enla ging acheids (8 o 9, Fig. 5) g ea ly di -
e ed om hose yielded in wall- hickening ones (1 o 7, Fig. 5).
Figu e 1. Theo e ical scheme o in aannual dynamics o xylogenesis in empe a e coni e s. Cu es p o ided in he li e a u e A) based on he a i-
a ion in he numbe o cells coun ed ac oss he 4 main zones (cambial, enla ging, wall hickening, and ma u e) and B) conside ing SCW deposi ion
and cell wall ligni ica ion as 2 sepa a ed di e en ia ion p ocesses. Backg ound boxes ep esen heo e ical ime pe iods o he co esponding
phenophases.
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Whe eas he angen ial walls in 8 and 9 lacked he spec oscopy
signa u e o lignin a 1,599 cm
−1
, an in ense signal was ob-
se ed in he RMS spec a in 1 o 7. The g ea es change in
he cellulose- o-lignin (C/L) a io (indica ing in ense ligni ica-
ion) wi hin he wall- hickening zone was eco ded be ween lo-
ca ions 5 and 6 (Fig. 6, Supplemen a y Fig. S3), while mos o he
a ia ion (48 ou o 93%) in he ela i e in ensi y o he RMS
sca e ing o he lignin band ac oss he en i e di e en ia ion
zone occu ed be ween loca ions 7 and 8 (Fig. 6). These esul s
indica ed ha lignin was deposi ed in he middle lamella well
be o e he end o SCW deposi ion. Indeed, he spec a mea-
su ed in loca ions 1 o 5 closely esembled hose o ma u e xy-
lem (compa e Fig. 2 wi h Fig. 5). Mo eo e , he limi ed change
in he C/L a io ( om 0.21 o 0.34) eco ded be ween ma u e
xylem and loca ions 1 o 5 poin ed ou ha middle lamella
ha dly ligni ied a e cell dea h o e en du ing la e s ages o
SCW deposi ion (Fig. 6). Changes in he ela i e signal in ensi y
wi hin he enla ging zone (loca ions 8 and 9) we e limi ed o a
mo e p ominen signal o he cellulose spec al band in loca-
ion 8 (Supplemen a y Fig. S3), indica ing an inc eased cellulose
con en in he PCW.
Changes in lignin deposi ion ac oss cell wall laye s
The ela i e signal in ensi y o he RMS spec a o he lignin
band and he au o luo escence emission exci ed a 405 nm
eco ded a he same loca ions wi hin he di e en ia ion
zone showed ema kably simila alues (Fig. 6). This was con-
i med by a linea eg ession be ween he 2 signals (R
2
= 0.91,
Fig. 6), poin ing ou a high co espondence be ween he
au o luo escence emission exci ed a 405 nm (blue colo in
Fig. 4) and lignin con en . Such co espondence enabled a de-
ailed analysis o lignin concen a ions ac oss cell wall laye s
based on highly esol ed CLSM images. The eby, CC and CML
we e ound o be mo e ligni ied han SCW, excep o he S3
laye , which was ich in lignin (Fig. 4, Supplemen a y Fig. S2).
Indeed, he S2 laye could be easily dis inguished om he S3
and CML hanks o i s lowe au o luo escence signal in en-
si y (Supplemen a y Fig. S2), being much hicke in la ewood
han in ea lywood acheids. Mo e in e es ingly, lignin ap-
pea ed o be p esen in middle lamella o s ill hin-walled
acheids (especially in CC), con i ming ha ligni ica ion
was unde way a e y ini ial s ages o SCW o ma ion
(Fig. 4). Al hough lignin was also p esen in he SCW in olde
wall- hickening cells, a lignin-de icien laye lined he inne
cell wall bounda y, e ealing a empo al o se be ween cellu-
lose mic o ib il deposi ion and lignin imp egna ion wi hin
he SCW (Fig. 4, bu see Supplemen a y Fig. S2 o highe de-
ail). The wid h o he lignin-de icien laye appea ed no o
a y ega dless o he conside able change in wall hickness
ac oss di e en ia ing acheids.
In aannual dynamics o wall hickening and
ligni ica ion a cell and issue le els
In all he s udied ees, he numbe o de eloping bi e in-
gen cells (as no ed h ough TLM) ma ched he numbe o
ligni ying cells coun ed using CLSM images (Fig. 7), e ealing
a co espondence be ween he onse o CML ligni ica ion
and SCW deposi ion. De ia ions be ween TLM and
CLSM-de i ed cu es we e educed, wi h he di e ence in
Figu e 2. RMS explo a ion o ma u e xylem. A) Au o luo escence image showing he spec al emission (415 o 691 nm) ollowing a single 405 nm
exci a ion on 32 colo ed channels using he lambda mode spec a o CLSM. B) Raman spec um eco ded on he middle lamella be ween 2 ma u e
acheids in he a ea delimi ed by a ci cle in he TLM image C). D) Raman chemical image (box in A and C) o he cell wall a ea based on he lignin
speci ic band a 1,599 cm
−1
(in eg a ed in ensi ies be ween 1,592 and 1,604 cm
−1
). Magni ica ion: 50× objec i e o he spec a and 80× objec i e
o he hype spec al map.
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he numbe o cells oscilla ing be ween 0 and 3 acheids
(1.96 ± 1.95 cells on a e age, Supplemen a y Table S1).
Cell-le el imings and du a ions p edic ed om indi idual
models (Supplemen a y Fig. S4) we e also ema kably coinci-
den (Fig. 8), wi h a sligh de ia ion only p esen o he las -
o med la ewood cells. A mean o se o only 0.96 d be ween
he 2 cu es poin ed ou he high synch onici y be ween wall
hickening (TLM) and ligni ica ion (CLSM) a he cell le el,
which was conse ed along he ee ing and ac oss ees des-
pi e he con as ing indi idual pa e ns (Fig. 7). On a e age,
acheids equi ed 25.7 (27.1) d o comple e wall hickening
(ligni ica ion), esul ing in a mean di e ence o 1.9 (SD = 5.5)
d. Maximal du a ions we e obse ed in la ewood (i.e. hey
ook 69 mo e d o be comple e in la ewood han in ea ly-
wood cells). A he issue le el, wall hickening (TLM) and lig-
ni ica ion (CLSM) anged o e he same pe iod, showing
coinciden onse (day o yea - DOY - 138 o 164) and end
(DOY 268 o 333) da es (Supplemen a y Table S1).
Discussion
Mul imodal imaging as a eliable app oach o assess
ligni ica ion in xylogenesis s udies
In his s udy, we p esen a comp ehensi e analysis o acheid
ligni ica ion in sil e i by combining spa ial in o ma ion de-
i ed om RMS and empo al dynamics econs uc ed om
TLM and CLSM images. RMS chemical imaging p o ides mo-
lecula insigh s in o cell wall composi ion by using unique
spec al inge p in s o iden i y compounds being pa icula -
ly use ul o lignin de ec ion (Gie linge e al. 2012; Guillon
e al. 2022; Leng e al. 2022). Howe e , he long ime equi ed
o ob ain and p ocess RMS spec a may limi a widesp ead
applica ion in xylogenesis s udies, which ypically equi e
obse a ions on nume ous samples (Ra hgebe e al. 2016).
P io s udies ha e success ully analyzed ligni ica ion by using
CLSM images (Dickson e al. 2017; Nanayakka a e al. 2019).
Ye , he associa ion be ween na i e wood luo escence and
lignin con en is no always s aigh o wa d. Al hough he
dis ibu ion pa e n o he signal exci ed by 405 nm ligh
(blue channel) was consis en wi h he expec ed o lignin,
an op imal lignin au o luo escence exci a ion would only
be achie ed by using an ul a iole (280 o 355 nm wa e-
leng h) lase (Dickson e al. 2017; Donaldson 2020). Indeed,
while some na u al phenolic compounds could luo esce a
simila wa eleng hs as lignin (Donaldson e al. 2019), a i icial
compounds (e.g. moun ing media) and changes in pH could
modi y na i e wood luo escence (Donaldson 2013; Ding
e al. 2020; Donaldson 2020). Fu he mo e, al hough luo es-
cen dyes could enhance lignin isualiza ion (Ki in e al. 2003,
2020; Balzano e al. 2021), i is impo an o exe cise cau ion
when in e p e ing esul s due o po en ial luo escence
quenching caused by subop imal dye concen a ions (Bond
e al. 2008). This mo i a ed us o es he co ela ion be ween
he ela i e in ensi y o he RMS band o lignin and he au o-
luo escence exci ed by iole /blue ligh (405 nm) in di e en-
ia ing xylem. The high co ela ion yielded be ween he 2
signals demons a es ha 405-nm ligh is sui able o exci e
lignin au o luo escence in sil e i (Fig. 6), alida ing he
use o ou CLSM images o moni o ligni ica ion. These e-
sul s unde sco e he po en ial o mul imodal imagining ap-
p oaches o eliably analyze xylogenesis.
Insigh s in o he ligni ica ion o sil e i acheids
Lignin emi ed he s onges RMS sca e ing and luo es-
cence signal wi hin ma u e xylem, al hough i s ela i e con-
cen a ion a ied ac oss cell wall laye s. CC and CML emi ed
Figu e 3. RMS explo a ion o he cambial zone. A) Au o luo escence image showing he spec al emission (415 o 691 nm) ollowing a single 405 nm
exci a ion on 32 colo ed channels using he lambda mode spec a o CLSM. B) A e age Raman spec um om 15 indi idual spec a eco ded wi hin
he egion o in e es (box in A) and C) TLM image including he Raman chemical image on he lignin-speci ic band a 1,599 cm
−1
(in eg a ed
in ensi ies be ween 1,592 and 1,604 cm
−1
). Magni ica ion: 50× objec i e.
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s onge luo escence han he S2 laye (Fig. 4,
Supplemen a y Fig. S2), sugges ing a highe lignin con en .
Limi ed image esolu ion challenged dis inc ion be ween
S1 and CML, which could also e lec a ela i ely simila lignin
con en in hese 2 laye s. Lowe lignin concen a ion in S2
han in CML is common in no mal coni e wood (Ku scha
and Schwa zmann 1975; Donaldson 2001; Schmi e al.
2003; Aga wal 2006). This may esul om highly condensed
lignin uni s o med in he CC and middle lamella due o hei
ich pec ic composi ion (Te ashima and Fukushima 1988;
Boe jan e al. 2003; Te ashima e al. 2004), bu also om as-
socia ions be ween lignin and he hemicelluloses ha in e -
cala e cellulose mic o ib ils in he PCW (Te ashima e al.
2004). Ye , RMS spec a showed a limi ed con ibu ion o
hemicelluloses in CML, which could be pa ly ela ed o hei
nonc ys alline s uc u e (Aga wal 1999; Gie linge e al.
2012). As epo ed in p e ious s udies (Donaldson 2001;
Aga wal 2006), he las o med laye (S3) e ealed a mo e in-
ense ligni ica ion han he S2 (Supplemen a y Fig. S2). Nea
S3 bounda ies in CLSM images e idenced ha S2 was much
hicke in la ewood han in ea lywood, which accoun s o
he dec ease in lignin concen a ion om ea lywood o la e-
wood walls p e iously epo ed in coni e s (An ono a e al.
2014). Beyond con i ming he eliabili y o ou obse a ions,
hese esul s sugges ha he lignin dis ibu ion in e ed
om ou analysis is pe asi e ac oss coni e species.
Topochemical in o ma ion ob ained h ough CLSM and
RMS images shed ligh on cell wall ligni ica ion in sil e i .
As expec ed, he spec oscopic signa u e o lignin p e ailed
in ma u e cells, while i was negligible in cambial and enla -
ging cells (Figs. 2, 3, and 5). Ini ial lignin- ela ed au o luo es-
cence in di e en ia ing acheids was obse ed in CC and
CML, sp eading la e inwa d in o he SCW. This aligns wi h
p e ious s udies showing ha ligni ica ion ini ia es a hese
speci ic si es (Ku scha and Schwa zmann 1975; Te ashima
and Fukushima 1988; Donaldson 1991; Fukushima and
Te ashima 1991; Donaldson 2001; Tobima su e al. 2013;
Meen s e al. 2018). The a iable lignin concen a ion ob-
se ed ac oss di e en ia ing cells sugges s dynamic changes
in lignin biosyn hesis a es du ing cell di e en ia ion. The
g ea es a ia ion in he ela i e lignin concen a ion o
CML was eco ded a ini ial wall- hickening s ages (loca ions
7 and 8, Fig. 6), which could be a ibu ed o highe ligni ica-
ion a es in middle lamella han in he S2 (Te ashima and
Fukushima 1988). Ligni ica ion a es would hen inc ease
again in he S3 (Donaldson 2001; Schmi e al. 2003;
G iča e al. 2005), as sugges ed in ou ees by he highe lig-
nin con en . He e ogeneous ligni ica ion ac oss cell wall
laye s would be accoun ed by sequen ial deposi ion o hemi-
celluloses and oxida i e enzymes (Meen s e al. 2018), which
a e esponsible o he polyme iza ion o monolignol depos-
i s sec e ed by Golgi esicles (Samuels e al. 2002). Ye , a g ad-
ual inc ease in CML lignin concen a ion un il ull cell
ma u a ion sugges ed a ce ain o e lap be ween CML and
SCW ligni ica ion, which could be a ibu ed o mobili y o
monolignols in he polysaccha ide ma ix (Meen s e al.
2018). Indeed, one p e ious s udy in balsam i (Abies balsamea
(L.) Mill.) epo ed an o e lap be ween bo h PCW and SCW
Figu e 4. TLM and CLSM images o ans e se wood mic osec ions. T ansmi ed ligh image o uns ained mic osec ion showing xylem and cambial
zone unde whi e ligh A) and pola ized ligh B). C) Composi e au o luo escence image o xylem, ascula cambium, and phloem showing he emis-
sion signal o di e en luo opho es exci ed simul aneously a 405 and 488 nm a e applying linea unmixing spec a. D) Composi e image o di -
e en ia ing xylem and co esponding g ay-scale images a e spli ing he signals exci ed a 405 nm E) and 488 nm F). Do ed line 1 in D o F)
highligh he ange o ligni ying cells along 1 adial ile, de ined by he ela i e dominance o he luo opho es exci ed a 405 and 488 nm. Boxe
2 in D o F) highligh he younges ligni ying cell, wi h luo escence exci ed a 405 nm being dominan in he middle lamella. Box 3 in D o F) high-
ligh he oldes ligni ying cell, wi h luo escence exci ed a 488 nm being dominan in he S3 laye .
Coo dina ion o cellulose and lignin deposi ion PLANT PHYSIOLOGY 2024: 195; 2428–2442 |2433
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ligni ica ion, bu also be ween he S3 and he wa y laye s
(Ku scha and Schwa zmann 1975; Schmi e al. 2003). These
esul s howe e con as wi h hose epo ed in pines, wi h
middle lamella being ully ligni ied be o e he s a o SCW lig-
ni ica ion (Donaldson 1991; Fukushima and Te ashima 1991;
Donaldson 2001). Thus, whe he a a iable o e lap in ligni ica-
ion be ween CML and SCW ansla es in o dis inc cell-le el
du a ions o ligni ica ion ac oss species is a ques ion ha de-
se es u he in es iga ion.
A e cell au olysis, ligni ica ion could con inue h ough e-
leases o acuola monolignol glucosides in o he cell wall
(Samuels e al. 2002; Pesque e al. 2013). Ye , he limi ed
C/L a io inc emen eco ded be ween wall- hickening and
ma u e acheids in CC and CML (Fig. 6) sugges s ha pos -
mo em ligni ica ion (i any) would be limi ed o SCW, pa -
icula ly S3 and wa y laye s (Schmi e al. 2003; G iča
e al. 2005). Mo eo e , pa enchyma and o he cell ypes
(e.g. epi helial cells) could become ligni ied a e he comple-
ion o he ee ing (Ku scha and Schwa zmann 1975;
Donaldson 2001), po en ially a ec ing he deg ee o ligni ica-
ion o nea by ma u e achea y elemen s (Pesque e al.
2013; Ba os e al. 2015). Al hough he con ibu ion o neigh-
bo ing cells o acheid ligni ica ion was no speci ically
quan i ied in ou samples, acheids adjacen o pa enchyma
ays exhibi ed a compa able deg ee o ligni ica ion o hose
loca ed a mo e dis an loca ions wi hin he same adial pos-
i ion (Fig. 4). This sugges ed ha he con ibu ion o neigh-
bo ing cells o ma u e acheid ligni ica ion would ha e a
limi ed quan i a i e impac .
Cell kine ics econs uc ed in ou s udy showed ha
acheids spen an a e age o 30 d in he ligni ica ion zone,
which is consis en wi h a p e ious s udy epo ing se e al
weeks o ligni ica ion wi hin a single cell (Sa idge and
Udagama-Randeniya 1992). Howe e , cell esidence imes
in he ligni ica ion zone inc eased along he ee ing, wi h
much longe pe iods o ligni ica ion in la ewood han in ea -
lywood (Fig. 8). Va iable ligni ica ion pe iods h oughou he
ee ing may a ise due o an inc emen in cell wall hickness
om ea ly o la ewood, bu also o seasonal luc ua ions in
lignin deposi ion a es. Acco ding o An ono a e al.
(2007), ligni ica ion is mo e in ense du ing la e s ages o
cell wall o ma ion in ea lywood acheids and du ing ea ly
s ages in la ewood acheids, which was a ibu ed o
changes in lignin p ecu so s and he ca bohyd a e ma ix
h oughou he g owing season. La ewood cells equi ing a
long ime o become ligni ied could also esul om dec eas-
ing empe a u es in he all (Donaldson 2001; G iča e al.
2005). Recen esea ch has highligh ed he signi icance o
empe a u e in con olling ligni ica ion (C i ella o and
Bün gen 2020), which could cease ab up ly a e
Figu e 5. RMS explo a ion o di e en ia ing xylem. A) Au o luo escence image showing he spec al emission (415 o 691 nm) ollowing a single
405 nm exci a ion on 32 colo ed channels using he lambda mode spec a o CLSM. Nine loca ions o spec a eco ding along 3 egions o in e es : 2
including wall- hickening acheids on he le (loca ions 1 o 7) and 1 including hin-walled enla ging acheids on he igh (loca ions 8 and 9).
B) A e age spec um om 4 indi idual spec a eco ded in loca ions 1 o 9, each a e aged o e an a ea o 4 pixels (pixel = 1 µm × 1 µm). Pu e
His olaque spec um is added o compa ison. C) Raman chemical images a e based on he lignin-speci ic band a 1,599 cm
−1
(in eg a ed in ensi ies
be ween 1,592 and 1,604 cm
−1
). Magni ica ion: 50× objec i e.
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excep ionally cold episodes (Schmi e al. 2003; Pie ma ei
e al. 2020), o being delayed un il sp ing unde mild win e
condi ions (Donaldson 1991; Nanayakka a e al. 2019).
Howe e , a ull unde s anding o he clima e d i e s o ligni i-
ca ion would equi e u he esea ch analyzing he in aann-
ual dynamics o ligni ica ion unde con as ing clima e
condi ions.
Coo dina ion be ween SCW deposi ion and cell wall
ligni ica ion
Ou second hypo hesis e e ed o he ime lags be ween
SCW deposi ion and cell wall ligni ica ion. A la ge empo al
o se be ween he 2 phases was no suppo ed by RMS
and CLSM images, as signi ican amoun s o lignin we e ob-
se ed in CC and middle lamella o acheids a he e y
Figu e 6. Rela i e lignin concen a ions ac oss di e en ia ing xylem. A) C/L a ios calcula ed om he ela i e in ensi y o he RMS sca e ing o
lignin and cellulose bands in ma u e (loca ion in Fig. 3B) and wall- hickening xylem (loca ions 1 o 7 in Fig. 5A). B) Compa ison o he ela i e
in ensi y o he Raman sca e ing o he lignin band and au o luo escence signal ela ed o lignin (exci ed a 405 nm) ac oss di e en ia ing xylem
(loca ions 1 o 9 in Fig. 5A). C) Linea eg ession showing high co espondence be ween Raman and au o luo escence signals. RMS imaging o lignin
a 1,592 o 1,604 cm
−1
and cellulose a 1085 o 1,105 cm
−1
(exci a ion lase : 532 nm). CLSM mapping o au o luo escence a 468 nm (exci a ion
lase : 405 nm). E o ba s ep esen he SD (sample size: 4 pixels).
Coo dina ion o cellulose and lignin deposi ion PLANT PHYSIOLOGY 2024: 195; 2428–2442 |2435
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ini ial s ages o SCW deposi ion (Fig. 6). This is in line wi h
p e ious obse a ions showing ha lignin deposi ion in he
CML occu s a an ea ly s age o xylem di e en ia ion
(Fukushima and Te ashima 1991), being concu en wi h
he syn hesis o a signi ican ac ion o he o al cell wall cel-
lulose (An ono a e al. 2007). This idea also inds suppo
om p e ious s udies analyzing ligni ica ion h ough immu-
noelec on mic oscopy (Ruel e al. 2002; Joseleau and Ruel
2006), which sugges ha lignin imp egna es he SCW as
soon as he cellulose mic o ib il ma ix is deposi ed. As p e-
iously desc ibed in sil e i (Schmi e al. 2003; G iča e al.
2005) and pine (Donaldson 1991), wall- hickening acheids
con ained a hin unligni ied laye adjacen o he lumen
(Fig. 4, Supplemen a y Fig. S2), e ealing ha cellulose depos-
i ion in he SCW s a s be o e i s ligni ica ion. The limi ed size
o his unligni ied laye hin s a a sho o se be ween
cellulose mic o ib il deposi ion and lignin imp egna ion.
Howe e , u he explo a ion would be needed o accu a ely
de e mine he ime lag be ween he comple ion o SCW
deposi ion and ligni ica ion.
Ou quan i a i e analysis based on cell coun s showed a e-
ma kable concu ence be ween TLM- and CLSM-de i ed cell
kine ics (Figs. 7 and 8), con i ming a simila onse o SCW de-
posi ion and ligni ica ion and e u ing ou second and hi d
hypo heses. A po en ial explana ion is ha ligni ica ion likely
begins in CC and CML a ound he ime when cellulose mic o-
ib ils s a o be added o he SCW. In addi ion, ou obse a-
ions sugges ed ha mos lignin may ha e al eady been
inco po a ed in o he SCW be o e i s comple ion.
The e o e, cellulose and lignin deposi ion would be highly
o e lapped a he cell le el. These indings align wi h esul s
om a p e ious s udy in Mon e ey pine (Pinus adia a
D. Don) showing ha lignin au o luo escence and wall hick-
ness ac oss he o ming ee ing a e highly co ela ed
(Nanayakka a e al. 2019). Simila inc eases in cell-le el du a-
ions along he g owing season o wall hickening (TLM) and
ligni ica ion (CLSM) would e lec he s ong co egula ion be-
ween SCW deposi ion and ligni ica ion poin ed ou in a i-
ous s udies using ansgenic plan s (e.g. Ruel e al. 2002).
While low-lignin ansgenic plan s we e ound o ha e high le-
els o cellulose ma ix diso ganiza ion leading o xylem de-
o ma ion (Ruel e al. 2002; Voelke e al. 2011), ad anced
ligni ica ion in ansgenic aspen dis up ed PCW ex ension
a ea ly s ages o cell enla gemen (G ünwald e al. 2002).
These lines o e idence sugges ha acheid de elopmen e-
qui es a ine coo dina ion be ween he di e en subp ocesses
o di e en ia ion. This no ion alludes o he ac ha di e en
phases o xylogenesis may no only be linked by hei empo al
sequence bu also by hei gene al egula o y mechanisms.
Implica ions o coo dina ed SCW deposi ion and cell
wall ligni ica ion o ee- ing science
Ou esul s con e ge on he idea ha SCW deposi ion and lig-
ni ica ion a e ime coo dina ed om he cell up o he en i e
ee- ing le el. Mo eo e , hey would imply ha mos o he
cellulose and lignin in a pa icula ee ing o ee- ing sec o
a e inco po a ed o e compa able ime windows, e en
hough a ce ain amoun o lignin could s ill be deposi ed
a e he end o SCW deposi ion. Despi e ou s udy conce n-
ing empe a e coni e s, he e a e sou ces o e idence sugges -
ing simila esul s in empe a e angiospe ms (P islan e al.
2009). Fo ins ance, o e lapped deposi ion imes o cellulose
and lignin would explain pa allel in a ing ca bon iso ope p o-
iles o cellulose and o al wood ound in beech (Helle and
Figu e 7. In aannual a ia ion in he numbe o wall- hickening and
ligni ying acheids. Wall- hickening acheids (ci cles) we e measu ed
in 5 ees unde a ansmi ed ligh mic oscope, and ligni ying acheids
( iangles) we e ob ained a e p ocessing au o luo escence images.
Symbols (do s and iangles) ep esen a e aged cell coun s o 3 adial
iles o each da e and phase.
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