ORIGINAL RESEARCH
published: 08 Feb ua y 2017
doi: 10.3389/ pls.2017.00126
F on ie s in Plan Science | www. on ie sin.o g 1Feb ua y 2017 | Volume 8 | A icle 126
Edi ed by:
Rüdige Simon,
Uni e si y o Düsseldo , Ge many
Re iewed by:
Ma g e M. Sau e ,
Uni e si y o Kiel, Ge many
Sa ah Liljeg en,
Uni e si y o Mississippi, USA
*Co espondence:
F ancisco R. Tadeo
[email p o ec ed]
†P esen Add ess:
Paz Me elo,
Eu opean Molecula Biology
Labo a o y, De elopmen al Biology
Uni , Heidelbe g, Ge many;
Ja ie Agus í,
Depa amen o de Desa ollo y Acción
Ho monal en Plan as, Ins i u o de
Biología Molecula y Celula de
Plan as, Uni e sidad Poli écnica de
Valencia-Consejo Supe io de
In es igaciones Cien í icas, Valencia,
Spain;
Vicen A bona,
Depa amen de Cièncias Ag à ies i
del Medi Na u al, Uni e si a Jaume I,
Cas elló de la Plana, Spain;
Leand o H. Es o nell,
Depa men o Plan Biology, Swedish
Uni e si y o Ag icul u al Science,
Uppsala BioCen e, Uppsala, Sweden
Special y sec ion:
This a icle was submi ed o
Plan Physiology,
a sec ion o he jou nal
F on ie s in Plan Science
Recei ed: 17 No embe 2016
Accep ed: 20 Janua y 2017
Published: 08 Feb ua y 2017
Ci a ion:
Me elo P, Agus í J, A bona V,
Cos a ML, Es o nell LH,
Gómez-Cadenas A, Coimb a S,
Gómez MD, Pé ez-Amado MA,
Domingo C, Talón M and Tadeo FR
(2017) Cell Wall Remodeling in
Abscission Zone Cells du ing
E hylene-P omo ed F ui Abscission in
Ci us. F on . Plan Sci. 8:126.
doi: 10.3389/ pls.2017.00126
Cell Wall Remodeling in Abscission
Zone Cells du ing E hylene-P omo ed
F ui Abscission in Ci us
Paz Me elo1 †, Ja ie Agus í1 †, Vicen A bona1 †, Má io L. Cos a2, Leand o H. Es o nell1 †,
Au elio Gómez-Cadenas3, Sil ia Coimb a2, Ma ía D. Gómez4, Miguel A. Pé ez-Amado 4,
Concha Domingo1, Manuel Talón1and F ancisco R. Tadeo1*
1Cen e de Genòmica, Ins i u Valencià d’ Ag à ies, València, Spain, 2Depa amen o de Biologia, Faculdade de Ciências,
Uni e sidade do Po o, Po o, Po ugal, 3Depa amen de Ciències Ag à ies i del Medi Na u al, Uni e si a Jaume I, Cas elló
de la Plana, Spain, 4Depa amen o de Desa ollo y Acción Ho monal en Plan as, Ins i u o de Biología Molecula y Celula de
Plan as, Uni e sidad Poli écnica de Valencia-Consejo Supe io de In es igaciones Cien í icas, Valencia, Spain
Abscission is a cell sepa a ion p ocess by which plan s can shed o gans such as ui s,
lea es, o lowe s. The p ocess akes place in speci ic loca ions e med abscission zones.
In ui c ops like ci us, ui abscission ep esen s a high pe cen age o annual yield
losses. Thus, unde s anding he molecula egula ion o abscission is o capi al ele ance
o con ol p oduc ion. To iden i y genes p e e en ially exp essed wi hin he ci us ui
abscission zone (AZ-C), we pe o med a compa a i e ansc ip omics assay a he cell
ype esolu ion le el be ween he AZ-C and adjacen ui ind cells (non-abscising issue)
du ing e hylene-p omo ed abscission. Ou s a egy combined lase mic odissec ion wi h
mic oa ay analysis. Cell wall modi ica ion- ela ed gene amilies displayed p ominen
ep esen a ion in he AZ-C. Phylogene ic analyses o such gene amilies e ealed a link
be ween phylogene ic p oximi y and exp ession pa e n du ing abscission sugges ing
highly conse ed oles o speci ic membe s o hese amilies in abscission. Ou
ansc ip omic da a was alida ed wi h (and s ongly suppo ed by) a pa allel app oach
consis ing on ana omical, his ochemical and biochemical analyses on he AZ-C du ing
ui abscission. Ou wo k iden i ies genes po en ially in ol ed in o gan abscission and
p o ides ele an da a o u u e bio echnology app oaches aimed a con olling such
c ucial p ocess o ci us yield.
Keywo ds: calyx abscission zone, cell wall modi ica ion, ci us ui abscission, e hylene, lignin biosyn hesis,
phylogeny, ansc ip omics
INTRODUCTION
Abscission is a cell sepa a ion p ocess by which plan s can shed hei ae ial o gans. I akes place
in g oups o unc ionally specialized cells known as abscission zones (AZs), which a e loca ed a
speci ic si es o o gan de achmen in he plan (Robe s e al., 2002; Es o nell e al., 2013; Tucke
and Kim, 2015).
Abscission is a undamen al p ocess in plan biology ha ep esen ed a highly bene icial
e olu iona y adap a ion o plan s: abscission allows o disca ding senescen o physiologically
damaged o gans and o highly e icien seed dispe sal. Howe e , om an ag icul u al poin o iew,
abscission has a emendous impac on yield, leading o high yield losses in key c ops like b assica
Me elo e al. Cell Wall Remodeling du ing Abscission
o ci us. In his way, unde s anding abscission a he molecula
le el is o op ele ance no only o unde s and a undamen al
p ocess o plan physiology bu also o gene a e new, imp o ed,
highly p oduc i e c ops.
Abscission ela ed ai s (i.e., educed abscission o ui s
o seeds) a e among he main ag onomic ai s selec ed along
plan domes ica ion (Konishi e al., 2006; Picke sgill, 2007). A
cu en example is he expansion o la e-season a ie ies o swee
o ange in he ci us ma ke . In such a ie ies, he decline in
he ui e en ion o ce is delayed du ing he ma u ing pe iod
in compa ison wi h ea ly and mid-season a ie ies (Gallasch,
1996) ha usually unde go p e-ha es ui abscission (Spiegel-
Roy and Goldschmid , 1996). Thus, la e-season a ie ies o swee
o ange ex end he ui ha es ing season bene i ing g owe s and
ood indus y.
Con ol o abscission is also ele an o acili a e mechanical
ha es ing, hus educing collec ion cos s. Mechanical ui
ha es ing sys ems ha e been de eloped al hough hey a e s ill
ine icien and cause ee damages (Li e al., 2011). On he
o he hand, se e al abscission- igge ing compounds ha e been
used o imp o e mechanical ha es ing. In ci us, ea men s
wi h CMNP (5-chlo o-3-me hyl-4-ni o-1H-py azole) a e used
o p omo e ui loosening and o acili a e and coo dina e
mechanical ha es ing o ui s (Bu ns, 2002). In his ega d,
unde s anding he mechanisms unde lying abscission is essen ial
o con ol abscission and imp o e ha es ing p ac ices and
p oduc i i y.
S udies on lo al o gan abscission in he model sys em
A abidopsis haliana ha e p o ided a weal h o aluable
in o ma ion. Howe e , he cu en in o ma ion abou he
molecula mechanisms unde lying abscission in c op species is
a he sca ce.
Mos o he molecula s udies o abscission in c ops ha e
mainly been ocused on he cha ac e iza ion o indi idual o
ew genes. Howe e , high- h oughpu app oaches ha e ecen ly
been applied in AZ-con aining issues o oma o lowe s (Mei
e al., 2010) and apple (Zhu e al., 2011), ma u e oli e (Gil-
Amado and Gomez-Jimenez, 2013; Pa a e al., 2013), melon
(Co bacho e al., 2013), li chi (Li e al., 2015), and o ange ui s
(Cheng e al., 2015). In ou p e ious s udies (Agus í e al., 2008,
2009, 2012), global exp ession analyses p o ided a wide se o
genes po en ially in ol ed in ci us lea abscission. These da ase s
included a numbe o cell wall modi ica ion ela ed genes as
well as genes in ol ed in signaling, ansc ip ion con ol, p o ein
syn hesis and deg ada ion and esicle anspo .
Ou cu en challenge is o iden i y key egula o y genes
o ci us ui abscission which is, indeed, an economically
impo an p ocess. In ci us, ma u ing ui s a e shed h ough
he abscission zone C (AZ-C), loca ed a he bounda y be ween
he calyx bu on and he ui ind (FR). In his egion, di e en
issues con e ge and he isola ion o exclusi e AZ-C cells o
molecula s udies wi hou any con amina ion o o he cell-
ypes is ex emely complica ed. In his s udy, we ha e aken
ad an age o he op imiza ion o lase mic odissec ion (LM) in
ci us issues (Agus í e al., 2009; Ma as e al., 2010; Ca uso
e al., 2012) o he accu a e sampling o ui AZ-C cells. This
s a egy has allowed he p ecise quan i ica ion o he iming
and magni ude o gene exp ession and associa e me aboli es
in ol ed in he p ocess o e hylene-p omo ed abscission in he
speci ic cells o he AZ-C. Mo eo e , phylogene ic analyses o he
mos ep esen a i e gene amilies du ing abscission in ci us and
di e en plan species ha e e ealed a link be ween phylogene ic
p oximi y and exp ession pa e n du ing his p ocess sugges ing
highly conse ed unc ions o speci ic membe s o hese amilies
in abscission. O e all, his s udy, h ough he iden i ica ion
o po en ial abscission- ela ed genes and he de ailed spa io-
empo al analysis o he ana omical and his ochemical changes
in he ac i a ed AZ-C, p o ides c ucial in o ma ion o u u e
bio echnological app oaches aimed a imp o ing ci us yield.
MATERIALS AND METHODS
Plan Ma e ial and In i o T ea men s
We used ui s om wo Ci us sinensis cul i a s: a mid-
season o ange cul i a (c . Washing on Na el) ha usually
unde goes p e-ha es abscission and a la e-season o ange
cul i a (c . Ricala e Na el) wi h delayed abscission. Ma u ing
ui s we e ha es ed a e colo change om adul ees g own
in a homogeneous expe imen al o cha d unde no mal cul u al
p ac ices a he Ins i u Valencià d’In es igacions Ag à ies
(IVIA). F ui s we e sepa a ed om he ee lea ing 2 cm
peduncles o isola e he AZ-C o u he analyses. Fo abscission
kine ics s udies and issue collec ion, Washing on Na el ui s
we e incuba ed o 0, 24, 48, and 96 h in he p esence o absence
o e hylene (10 µL/L) in sealed 10 l con aine s a 22◦C wi h a
16 h ligh pe iod unde luo escen ligh ing. Ricala e Na el ui s
we e incuba ed o 0, 24, 48, 96, and 192 h in he p esence o 1-
aminocyclop opane-l-ca boxylic acid (ACC; 0.1 mM) o wa e
unde he same empe a u e and ligh condi ions. In his case,
a 3 mL Pas eu pipe e con aining he ACC solu ion o wa e was
i ed o he ui peduncles.
Phlo oglucinol S aining
Phlo oglucinol s aining o lignin in esh cu issue po ions (0.5
cm3) con aining he AZ-C a e 0, 24, and 48 h o e hylene o
ACC ea men was pe o med acco ding o Tadeo and P imo-
Millo (1990). Samples we e cu longi udinally o allow AZ-C
s aining and o u he image acquisi ion. A sa u a ed solu ion o
phlo oglucinol (Sigma-Ald ich) in 20% HCl was di ec ly applied
o samples. Obse a ion was ca ied ou wi h an Olympus SZ61
s e eomic oscope (Olympus GmbH).
C yoscanning Elec on Mic oscopy
(c yo-SEM)
Longi udinal sec ions as well as he p oximal (peduncle) and
dis al ( ui ) ac u e plane o he e hylene-p omo ed AZ-C we e
obse ed using c yo-SEM. To examine longi udinal sec ions o
he AZ-C, 1 cm po ions o issue we e manually dissec ed
wi h a azo blade. In he second case, he peduncle was
o cibly sepa a ed om he ui . Specimen moun ing and AZ-
C obse a ion we e ca ied ou as p e iously desc ibed in Agus í
e al. (2009). A leas h ee samples con aining he AZ-C a e 24,
48, and 96 h o e hylene ea men we e obse ed.
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Me elo e al. Cell Wall Remodeling du ing Abscission
Pe iodic Acid-Schi (PAS) S aining
Tissue con aining he AZ-C a e 0, 24, and 48 h o ACC
ea men was manually dissec ed using a azo blade in 0.5
cm3po ions. These samples we e ixed o e nigh a 4◦C in a
4% (w/ ) pa a o maldehyde-PBS solu ion. A e ixa ion, samples
we e washed wi h PBS, dehyd a ed in a g aded e hanol se ies
and embedded in LR Whi e (Elec on Mic oscopy Sciences).
Longi udinal sec ions o he calyx bu on a ea (1 µm hickness)
we e cu wi h a Leica RM2165 mic o ome and placed on glass
slides. Slides we e u he s ained wi h PAS (Sigma-Ald ich)
and moun ed wi h DPX Moun an (Fluka). Obse a ions we e
pe o med on a Leica DMLA mic oscope (Leica Mic osys ems)
and images we e p ocessed wi h Leica ASMLD Ve sion 4.0
so wa e.
P epa a ion o Tissue Con aining he AZ-C
o LM
Po ions o issue con aining he AZ-C (0.5 cm3) we e dissec ed
om ui s a e 0, 12, and 24 h o e hylene ea men o he
ansc ip omics assay, and a e 0, 12, 24, and 36 h o ACC
ea men o lignin in e media es quan i ica ion. P epa a ion o
c yosec ions and mic odissec ion we e pe o med as p e iously
desc ibed in Agus í e al. (2009). Cells om he AZ-C and
he adjacen FR we e selec ed om 30 o 40 c yosec ions and
collec ed sepa a ely.
Phlo oglucinol S aining o C yosec ions
C yosec ions o 14 µm o issue con aining he AZ-C a e 48
h o e hylene ea men we e p ocessed as desc ibed in Agus í
e al. (2009) and moun ed on C yoJane R
adhesi e coa ed slides
(Ins umedics) ollowing he manu ac u e ’s ins uc ions. Slides
we e s o ed a −80◦C un il phlo oglucinol s aining. S aining o
lignin was pe o med using a sa u a ed solu ion o phlo oglucinol
(Sigma-Ald ich) in 20% HCl. Obse a ion was ca ied ou wi h
an Olympus SZ61 s e eomic oscope (Olympus GmbH).
RNA Isola ion, Sample Labeling, and
Mic oa ay Hyb idiza ion
Th ee independen biological eplica es we e collec ed o
each cell ype a 0, 12, and 24 h a e e hylene ea men . Fo
each independen sample, o al RNA om ∼40,000 pooled
cells was ex ac ed using he RNeasy Mic o Ki (Qiagen)
ollowing he manu ac u e ’s ins uc ions. The RNA pu i y
was assessed by measu emen s o OD260/OD280. Two RNA
ampli ica ion ounds we e pe o med u ilizing he Ta ge AmpTM
2-Round Aminoallyl-aRNA Ampli ica ion Ki (EPICENTER)
acco ding o he manu ac u e ’s ins uc ions o syn hesize
he an isense cRNA. The quali y o he ampli ied RNA was
e alua ed by OD260/OD280 measu emen s and aga ose gel
elec opho esis. Each sample was labeled wi h Cy5 and co-
hyb idized wi h Cy3-labeled an isense cRNA om a e e ence
sample con aining a mix u e o equal amoun s o RNA om all
expe imen al samples (0, 6, 12, 24, 48, and 96 h o e hylene/ai
ea men ). RNA labeling, mic oa ay hyb idiza ion, and slide
washes we e pe o med as p e iously desc ibed in Ce cos
e al. (2006). Hyb idized mic oa ays scanning, hyb idiza ion
da a acquisi ion, and mic oa ay no maliza ion and analysis
we e ca ied ou as desc ibed in Agus í e al. (2009). A cDNA
mic oa ay including 21.081 pu a i e genes o ci us was u ilized
(Ma inez-Godoy e al., 2008). Gene exp ession di e ences
we e conside ed signi ican unde a P- alue lowe han 0.05
and an M con as cu o alue o +0.5 o −0.5. In his wo k,
a ime cou se expe imen was designed o each cell ype
(AZ-C and FR), he e o e, he exp ession le el co esponds o
M=log2[AZ-C /AZ-C0] o M =log2[FR /FR0]. The aw
mic oa ay da a as well as he p o ocols used o p oduce he da a
and he no malized da a we e deposi ed in he A ayExp ess
da abase unde he accession numbe E-MTAB-4538. Func ional
classi ica ion o he selec ed genes was pe o med using
MIPS (Munich In o ma ion Cen e o P o ein Sequences,
h p://www.helmhol zmuenchen.de/en/mips/) ca ego iza ion.
Ampli ied RNA was used o he alida ion o mic oa ay
hyb idiza ion da a by semi-quan i a i e RT-PCR (sqRT-PCR)
analysis (Figu e S1).
sqRT-PCR Analysis
sqRT-PCR analysis was ca ied ou using he Supe Sc ip II
Re e se T ansc ip ase ki (In i ogen, Ca lsbad) ollowing he
manu ac u e ’s ins uc ions. A e i s -s and cDNA syn hesis,
PCR eac ions we e pe o med using he Bio ools Taq DNA
Polyme ase (BIOTOOLS, B&M Labs). Size and in ensi y o
expec ed bands we e checked by 1% aga ose gel elec opho esis.
Ci us UBC gene (Ubiqui in-conjuga ing enzyme) was used as
a e e ence o e alua e he amoun s o mRNA in each sample.
P ime sequences a e a ailable in Table S1.
In si u Hyb idiza ion
RNA in si u hyb idiza ion wi h digoxigenin-labeled p obes was
pe o med as desc ibed (Gomez e al., 2011). Po ions o issue
con aining he AZ-C (0.5 cm3) we e dissec ed om ui s
a e 24 h o e hylene ea men and immedia ely ixed a 4◦C
o e nigh in FAE (25% o maldehyde, 5% ace ic acid, 50%
e hanol), dehyd a ed, embedded in pa a in wax and sec ioned
o 8 µm. Fo Ci CEL6 and Ci PG20, RNA an isense and sense
p obes we e gene a ed wi h SP6 and T7 RNA polyme ases, using
as subs a e a 1518 bp agmen o he Ci CEL6 cDNA (1–1518
om ATG) o a 1110 bp agmen o he Ci PG20 cDNA (217–
1326 om ATG), ampli ied by PCR and cloned in o he pGEM-T
Easy ec o (P omega).
Lignin In e media es Quan i ica ion
Couma ic acid, ca eic acid, and e ulic acid we e analyzed by
UPLC coupled o andem mass spec ome y (UPLC-MS/MS)
as desc ibed by A gamasilla e al. (2014). Th ee independen
samples con aining ∼40,000 pooled AZ-C cells we e isola ed by
LM o each ime poin o ACC ea men (0, 12, 24, and 36 h)
and collec ed in 60 µL o wa e .
Sequence Iden i ica ion, Alignmen , and
Phylogene ic Analysis
Membe s o he di e en gene amilies associa ed wi h cell
wall emodeling in ci us (based on CAZy classi ica ion;
Ca bohyd a e-Ac i e Enzymes; Can a el e al., 2009;
h p://www.cazy.o g/) we e iden i ied by TBLASTN sea ch
F on ie s in Plan Science | www. on ie sin.o g 3Feb ua y 2017 | Volume 8 | A icle 126
Me elo e al. Cell Wall Remodeling du ing Abscission
in he Ci us clemen ina haploid genome ( e sion 0.9) da abase
web b owse (h p://www.phy ozome.ne /sea ch.php) using he
consensus sequence o he ca aly ic domain o each amily.
P edic ion o cha ac e is ic domains and conse ed mo i s was
ca ied ou h ough SMART (h p://sma .embl-heidelbe g.de/;
Schul z e al., 1998; Le unic e al., 2009), PSORT
(h p://pso .hgc.jp/ o m.h ml), In e P oScan
(h p://www.ebi.ac.uk/Tools/p a/ip scan/) and big-PI Plan
P edic o (h p://mendel.imp.ac.a /gpi/plan _se e .h ml)
se e s. Phylogene ic ees a e based on mul iple alignmen s
using he p o ile alignmen unc ion o Clus alW
(www.ch.embne .o g/so wa e/Clus alW-XXL.h m) and we e
gene a ed wi h MEGA7 (Kuma e al., 2016) using he neighbo -
joining algo i hm wi h 1000 boo s ap eplica es. Poisson
co ec ion o mul iple subs i u ions was used and only alues
highe han 50% we e conside ed.
Immunolocaliza ion o Pec ic
Polyssacha ides
The p ima y monoclonal an ibodies (mAbs) used in his
s udy and p o ided by P o . Paul Knox (Cen e o Plan
Sciences, Facul y o Biological Sciences, Uni e si y o Leeds,
UK) we e LM5 [an i-(1,4)-β-D-galac an; Jones e al., 1997],
LM6 [an i-(1,5)-α-L-a abinan; Willa s e al., 1998] and JIM5
[an i-pa ially me hyles e i ied/de-es e i ied homogalac u onan;
(Knox e al., 1990)]. The seconda y an ibody was luo escein
iso hiocyana e (FITC)-conjuga ed an i- a IgG (Sigma-Ald ich).
Immunolocaliza ion o pec ic polysaccha ides was pe o med on
semi- hin sec ions o issue con aining he AZ-C (0.5 cm3) om
ui s a e 0, 24, and 48 h o ACC ea men . Immunolocaliza ion
o pec ic polyssacha ides, ligh mic oscopy and image acquisi ion
we e pe omed as desc ibed in Coimb a e al. (2007).
RESULTS AND DISCUSSION
E hylene Accele a es Ci us F ui
Abscission
We pe o med a kine ics assay o ci us ui abscission in
esponse o abscission-accele a ing ea men s o de e mine he
op imal sampling o he ansc ip omic analysis. To ha end,
we ca ied ou a compa ison be ween o ange (C. sinensis)
ui s incuba ed wi h e hylene o i s immedia e me abolic
p ecu so 1-aminocyclop opane-1-ca boxylic acid (ACC) and
ui s incuba ed wi h ai o wa e (con ols). We used ma u ing
ui s om a mid-season o ange cul i a (c . Washing on
Na el) ha usually unde goes p e-ha es abscission and om
a la e-season o ange cul i a (c . Ricala e Na el) wi h delayed
abscission. We obse ed a as e dec ease o ui de achmen
o ce (FDF) in bo h Washing on Na el and Ricala e Na el
ui s ea ed wi h e hylene/ACC in compa ison o ai -/wa e -
ea ed con ol ui s (Figu e 1A). A 48 h a e ea men , he
FDF in ui s ea ed wi h e hylene o ACC was a ound 4 kg .
Howe e , con ol ui s o Washing on Na el and Ricala e Na el
only showed alues o FDF a ound 4 kg a 96 and 192 h,
espec i ely, a esponse ha ma ches hei p e-ha es abscission
beha io . Thus, e hylene and ACC accele a ed he abscission
FIGURE 1 | E ec o e hylene and 1-aminocyclop opane- 1-ca boxylic
acid (ACC) on ci us ui abscission. (A) Abscission kine ics o Washing on
Na el ui s non- ea ed o ea ed wi h e hylene and Ricala e Na el ui s
non- ea ed o ea ed wi h ACC. The esul s a e means o 10 ui s ±SE.
(B,C) Phlo oglucinol s aining o lignin in he AZ-C o Washing on Na el ui s
a e e hylene ea men (B) and Ricala e Na el ui s a e ACC ea men (C).
Dashed line, abscission zone C; , lignin deposi ion (phlo oglucinol); FR, ui
ind; FD, lo al disc; SP, sepals; VB, ascula bundles; P, pa enchyma.
p ocess in bo h a ie ies es ed. This esul s ongly sugges s ha
he na u al delay in he schedule o FDF decline in ui s o
he la e-season a ie y Ricala e Na el in compa ison wi h hose
o he mid-season a ie y Washing on Na el was no ela ed
o any impai men in he esponse o well-de eloped issues
o e hylene (Zaca ias e al., 1993). Based on hese indings, we
used induced AZ-C samples om bo h a ie ies o u he
analyses.
Phlo oglucinol S aining Re eals a Posi i e
Co ela ion be ween Abscission and Lignin
Deposi ion
Phlo oglucinol s aining in ecep acles o bo h Washing on Na el
and Ricala e Na el ui s e ealed lignin deposi ion a he cen al
co e o he AZ-C, be ween he axial ascula bundles, 24 h a e
e hylene and ACC ea men s (Figu es 1B,C). Fo y-eigh hou s
a e he ea men s, lignin deposi ion sp ead ou along he AZ-C,
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Me elo e al. Cell Wall Remodeling du ing Abscission
FIGURE 2 | Cellula mo phology o he AZ-C. (A–E) Scanning elec on mic og aphs o longi udinal sec ions (A,B,D) and he p oximal (C; calyx bu on side) and
dis al (E; ui ind side) ac u e planes o he AZ-C om Washing on Na el ui s a e 48 h (A,B) and 96 h (C–E) o e hylene ea men . High magni ica ion pic u es
show cells o sepa a ion laye s. AZ-C, abscission zone C; , sepa a ion line inside he AZ-C; CB, calyx bu on; FD, lo al disc; FR, ui ind; SP, sepal; VB, ascula
bundles. Scale ba s: 1 mm (A–E), 500 µm(A–C), 200 µm(E), 100 µm(C).
F on ie s in Plan Science | www. on ie sin.o g 5Feb ua y 2017 | Volume 8 | A icle 126

Me elo e al. Cell Wall Remodeling du ing Abscission
FIGURE 3 | Ana omy o he AZ-C. Pe iodic acid-Schi eac i e (PAS) s aining o insoluble ca bohyd a es o longi udinal sec ions o he AZ-C om Ricala e Na el
ui s non- ea ed (A,D) and ea ed o 24 h (B,E) and 48 h (C,F) wi h ACC. Squa es in (A–C) show he a ea magni ied wi h he 40X objec i e. AZ-C, abscission zone
C; FR, ui ind; DCA, di ided cells a ea; SA, s a ch- ich a ea; , ecen ly di ided cell; , cell con aining amyloplas s. Scale ba s: 500 µm and 50 µm.
pe ec ly d awing he sepa a ion line be ween he calyx bu on
and he FR. Acco dingly, iming o lignin deposi ion posi i ely
co ela ed wi h abscission kine ics.
Mo phological Changes in Ac i a ed AZ-C
Cells
We used scanning elec on mic oscopy (SEM) o examine
changes in he cellula mo phology o he AZ-C om
Washing on Na el ui s ea ed wi h e hylene (Figu e 2). We
obse ed he i s cellula signs o ac i a ion o abscission by
e hylene in he cen al co e o he AZ-C a 48 h o ea men
(Figu es 2A,B). A ha ime, AZ-C samples could be spli in o
wo g oups, one showing ea ly s ages o cell sepa a ion and he
o he one showing la e e en s o cell sepa a ion. In he o me
g oup, he AZ-C was clea ly dis inguishable (Figu e 2A), wi h
accumula ion o an amo phous ma e ial p obably de i ed om
he pa ial dissolu ion o he middle lamella and cell wall o he
AZ-C cells. In he la e g oup o samples, cell sepa a ion was
obse ed in he cen al co e o he AZ-C (Figu e 2B). A g ea e
accumula ion o amo phous ma e ial was obse ed, sugges ing
ha cell wall and middle lamella deg ada ion was comple e a e
48 h a he cen al egion. A 96 h a e e hylene ea men , cell
sepa a ion ex ended om he cen al co e o he pe iphe y o he
AZ-C (Figu e 2D) and di e en ial cell expansion was obse ed a
p oximal (calyx bu on) and dis al ( ui ) sides (Figu es 2C,E). A
he p oximal side, pa enchyma ic pi h cells unde wen expansion
(Figu e 2C) while, a he dis al side, expansion occu ed in he
cells o he axial ascula bundles (Figu e 2E).
Two Di e en Cell A eas Fo m he AZ-C
Pe iodic acid-Schi (PAS) s aining was used o cha ac e ize
ana omically he AZ-C a e ACC ea men (Figu e 3). This
me hod de ec s insoluble ca bohyd a es and was used o
dis inguish he cells belonging o he AZ-C since hese
accumula e s a ch g ains (Wilson and Hende sho , 1968;
Hube man e al., 1983; Shi aishi and Yanagisawa, 1988; Go en,
1993). In addi ion o he s a ch- ich cell a ea (SA) p e iously
iden i ied by Wilson and Hende sho (1968) a he dis al side o
he AZ-C (FR side), we iden i ied ano he cell a ea loca ed a he
p oximal side o he AZ-C (pi h side) and composed by ecen ly
di ided cells based on he obse a ion o hinne cell walls o med
be ween cells (Di ided Cells A ea, DCA; Figu e 3D). Then,
he AZ-C was cons i u ed by 10–15 cell laye s dis ibu ed in
cellula a eas wi h wo di e en cell mo phologies and o ganella
composi ion (i.e., cells om he SA con ain amyloplas s). The
analysis o he AZ-C a e 48 h o ea men sugges s ha
cell wall deg ada ion and cell degene a ion occu ed mainly
a he laye s o he SA in he ac u e plane, adjacen o he
mesoca p cells o he FR known as he albedo (see Figu e 9).
Howe e , cell expansion occu ed a he DCA (Figu e 3F).
These esul s oge he wi h kine ics (Figu e 1A), phlo oglucinol
s aining (Figu es 1B,C) and SEM (Figu e 2) da a sugges ed ha
he ac i a ion o he ui AZ-C by e hylene/ACC occu ed ea ly
a e ea men , p obably p io o 24 h. The e en s ela ed o
cell wall loosening migh s a a 24 h, while cell sepa a ion
seemed o begin a 48 h and o be comple ed a 96 h a e
ea men .
Gene Exp ession Regula ed by E hylene in
AZ-C and FR Cells
Fo gene exp ession analysis, we used he 20 K ci us cDNA
mic oa ay (Ma inez-Godoy e al., 2008). We isola ed cells om
he cen al co e o he AZ-C as well as om he FR loca ed
benea h he AZ-C h ough LM (Figu e S2) o pe o m a ime-
cou se expe imen (0, 12, and 24 h-e hylene) and compa ed da a
om each analysis. Resul s showed ha e hylene di e en ially
egula ed 2280 genes exclusi ely in he AZ-C cells, 1742 genes
exclusi ely in he FR cells, and 2001 genes we e egula ed by
e hylene in bo h he AZ-C and he FR cells (Table S1).
All di e en ially egula ed genes we e g ouped in o
unc ional ca ego ies acco ding o he Munich In o ma ion
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Me elo e al. Cell Wall Remodeling du ing Abscission
TABLE 1 | Rela i e gene exp ession alues (AZ-C s. AZ-C0) o genes in ol ed in cell wall modi ica ion exclusi ely egula ed by e hylene in AZ-C cells.
Name Con ig/single on ID Mic oa ay p obe Pu a i e A h o hologue Rela i e exp ession [log2 (AZ-C /AZ-C0)]
12 h 24 h
ENDO-1,4-β-GLUCANASES|CELLULASES (GH9s)
Ci CEL3 aCL1687Con ig1 IC0AAA38AD03 AT2G32990 – 0.69
Ci CEL6* aCL1347Con ig1 C21007H10 AT4G02290 – 4.15
Ci CEL10 aCL7597Con ig1 IC0AAA68DE06 AT4G02290 – 0.60
Ci CEL17 aCL1288Con ig1 C32011E04 AT1G75680 −0.88 –
Ci CEL22 aC20010F01SK_c C20010F01 AT1G23210 – 0.72
POLYGALACTURONASES (GH28s)
Ci PG6 aCL2029Con ig1 C03009E03 AT4G23820 –1.58 –
Ci PG16 aCL5261Con ig1 C01018A12 AT3G61490 1.02 –
Ci PG20* AT3G07970 in-si u
hyb idiza ion
Ci PG41 aC18008C05R _c C18008C05 AT3G57790 1.10 1.49
aCL1063Con ig1 IC0AAA19CA02 AT3G57790 0.88 0.95
Ci PG42 aIC0AAA85AB02RM1_c IC0AAA85AB02 AT3G48950 – 0.63
Ci PG43 aCL675Con ig4 IC0AAA67DG09 AT2G43870 – 2.34
PECTATE-LYASES (PL1s)
Ci PL1 aC03011D06SK_c C03011D06 AT5G63180 −0.86 −0.80
Ci PL5 aIC0AAA15AF11RM1_c IC0AAA15AF11 AT1G67750 2.66
PECTIN-METHYLESTERASES (CE8s)
Ci PME8 aC05807A09SK_c C05807A09 AT4G33220 −3.13 −2.57
Ci PME11 aCL1691Con ig1 C08033H07 AT1G11580 −2.30 –
Ci PME13 aCL4116Con ig2 C01011H09 AT5G53370 −2.15 −2.90
Ci PME24 aCL1451Con ig1 IC0AAA40DF03 AT1G69940 0.71 0.71
Ci PME41 aCL2379Con ig1 C32102B03 AT5G09760 – 1.41
PECTIN-ACETYLESTERASES (CE18s)
Ci PAE1 aKN0AAP13YN19FM1_c KN0AAP13YN19 AT3G62060 −0.72 –
Ci PAE4 aCL67Con ig4 C08028G04 AT4G19420 0.58 0.51
Ci PAE6 aCL7344Con ig1 C02003B05 AT5G26670 -1.57 –
aKN0AAI1DH10FM2_c KN0AAI1DH10 AT5G26670 −2.07 –
β-GALACTOSIDASES (GH35s)
Ci GBAL16 aC31805H10EF_c C31805H10 AT4G36360 −1.59
aCL7104Con ig1 C02004B02 AT4G36360 −1.01
β-GALACTOSIDASES (GH2s)
Ci GH22 aCL4443Con ig1 C31401H10 AT3G54440 0.71 0.95
β-GLUCOSIDASES (GH1s)
Ci BGLU17 aCL5744Con ig1 C31007D10 AT2G44480 −0.59 −0.55
Ci BGLU24 aCL1136Con ig3 IC0AAA1CB06 AT3G06510 – 0.62
β-MANNOSIDASES (GH5s)
Ci MAN4 aC04002G09SK_c C04002G09 AT1G02310 1.41 –
XYLOGLUCAN ENDOTRANSGLYCOSYLASES/HYDROLASES (GH16s)
Ci XTH16 aC02023G10SK_c C02023G10 AT4G03210 −1.55 −2.33
Ci XTH24 aIC0AAA99CH05RM1_c IC0AAA99CH05 AT1G32170 1.07 1.03
Ci XTH28 aCL6772Con ig1 C01009B04 AT4G37800 – −0.88
α-XYLOSIDASES (GH31s)
Ci XYL4 aCL6235Con ig1 C05075C11 AT1G68560 −0.98 –
β-XYLOSIDASES (GH3s)
Ci BXL13 aCL3345Con ig1 C02024D10 AT1G78060 −0.63 –
Ci BXL16 aCL8110Con ig1 IC0AAA75AA10 AT5G20950 −0.99 –
EXPANSINS
Ci EXP14 aCL2131Con ig1 IC0AAA14BD04 AT2G40610 – 3.01
aIC0AAA87BH09RM1_c IC0AAA87BH09 AT2G40610 1.63 3.19
(Con inued)
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Me elo e al. Cell Wall Remodeling du ing Abscission
TABLE 1 | Con inued
Name Con ig/single on ID Mic oa ay p obe Pu a i e A h o hologue Rela i e exp ession [log2 (AZ-C /AZ-C0)]
12 h 24 h
Ci EXP15 aKN0AAQ1YG09RM1_c KN0AAQ1YG09 AT4G17030 1.69 1.40
Ci EXP19 aC02006G07SK_c C02006G07 AT1G20190 – 0.75
CELLULOSE SYNTHASES/CELLULOSE SYNTHASE-LIKE PROTEINS
Ci Ces1 aC16012C03SK_c C16012C03 AT4G24010 1.82 –
Ci Ces2 aIC0AAA5DG11RM1_c IC0AAA5DG11 AT5G05170 -1.74 –
Ci Csl3 aCL5293Con ig1 C05070F01 AT3G03050 – 0.68
UDP-GLUCOSE 4-EPIMERASE
Ci UGE1 aC31108G08EF_c C31108G08 AT4G10960 – 0.95
Ci UGE2 aCL6604Con ig1 C03009D04 AT1G12780 – 0.82
MANNAN SYNTHASES
Ci ManS1 aCL3377Con ig1 IC0AAA16BH03 AT5G22740 – 1.47
Ci ManS2 aCL3377Con ig2 IC0AAA99AD02 AT5G22740 – 1.36
GALACTOMANNAN GALACTOSYLTRANSFERASE (GMGT)
Ci GMGT1 aC34004D03EF_c IC0AAA58DH01 AT2G22900 – 1.35
RHAMNOSE BIOSYNTHETIC ENZYME
Ci RHM1 aCL4478Con ig1 KN0AAI3AD11 AT1G78570 −0.52 –
GLUCOSYLTRANSFERASES
Ci GTF1 aCL3010Con ig2 IC0AAA58BE08 AT1G77130 0.82 –
Ci GTF2 aCL1592Con ig1 IC0AAA30DE02 AT1G16570 0.58 –
Ci GTF3 aCL3054Con ig2 KN0AAK3DE03 AT3G50060 – 1.21
Ci GTF4 aCL6931Con ig1 IC0AAA42BE09 AT3G25140 – 0.65
Ci GTF5 aCL5570Con ig1 C05056H08 AT1G77990 −1.42 −1.51
Ci GTF6 aCL6758Con ig1 C31701H10 AT3G02100 – −1.23
Ci GTF7 aKN0AAB3DB09ZM1_c KN0AAB3DB09 AT3G45400 – 0.60
GDP-L-FUCOSE SYNTHASE
Ci GLFS1 aCL790Con ig1 IC0AAA85AB07 AT1G17890 – 0.73
GALACTOSIDE 2-ALPHA-L-FUCOSYLTRANSFERASE
Ci GLFT1 aIC0AAA69BA06RM1_c IC0AAA69BA06 AT1G74420 – 0.58
Ci GLFT2 aCL5210Con ig1 C08029G10 AT1G05575 −0.87 −1.24
UDP-GLUCOSE DEHYDROGENASE
Ci UGD1 aKN0AAP5YD20FM1_c KN0AAP5YD20 AT5G15490 – 0.51
GLUCURONOSYL TRANSFERASE-LIKE PROTEIN
Ci GluT1 aCL8573Con ig1 C02015B05 AT3G55700 – −0.54
UDP-GLUCURONATE DECARBOXYLASE
Ci UGluD1 aCL1799Con ig2 C02011A11 AT2G28760 1.67 1.57
MANNOSYLTRANSFERASE-LIKE PROTEIN
Ci ManT1 aIC0AAA25BC01RM1_c IC0AAA25BC01 AT2G27100 0.77 0.60
–, No signi ican egula ion. (*) Localiza ion o gene exp ession in AZ-C cells by in-si u hyb idiza ion. Pu a i e gene iden i ica ions a e based on sequence homology wi h A abidopsis
haliana. Addi ional da a a e shown in Figu es S3–S6.
Cen e o P o ein Sequences (MIPS). The ca ego ies
suga , glucoside, polyol, and ca boxyla e me abolism and
polysaccha ide me abolism showed a highe pe cen age
o egula ion in he AZ-C compa ed wi h he FR
(Table S2).
The se o genes discussed below was selec ed because o
i s p ominen ep esen a ion in he AZ-C o i s pa icula
biological in e es . These gene amilies included hose associa ed
wi h cell wall me abolism and monolignol biosyn hesis and
polyme iza ion (Table S3).
Many Genes Rela ed o Cell Wall
Modi ica ion A e Regula ed du ing F ui
Abscission
A high numbe o genes encoding cell wall modi ica ion enzymes
we e di e en ially egula ed by e hylene exclusi ely in he ui
AZ-C. Ou da a sugges ed ha his s ong ac i a ion o cell wall
me abolism occu ed h ough bo h deg ada ion and biosyn hesis
(Table 1) as we p e iously showed in he lamina abscission
zone (LAZ) du ing lea abscission (Agus í e al., 2008, 2009,
2012). Genes encoding enzymes ha hyd olyze he cell wall and
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Me elo e al. Cell Wall Remodeling du ing Abscission
FIGURE 4 | Phylogene ic ela ionships be ween cellulases/endo-1,4-β-glucanases (CELs) and gene exp ession changes in esponse o e hylene
inAZ-C and FR cells. (A) CELs anno a ed in he Ci us clemen ina haploid genome (Wu e al., 2014), egula ed by e hylene in AZ-C cells and/o FR cells o
Washing on Na el ma u ing ui s and p e iously desc ibed as ela ed o he abscission p ocess in o he plan species a e shown. Phylogene ic ees a e based on
(Con inued)
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Me elo e al. Cell Wall Remodeling du ing Abscission
FIGURE 9 | Speci ic cellula and molecula e en s in ol ed in he dissolu ion o he middle lamella, he disassembly o cell walls and he syn hesis and
deposi ion o lignin in he AZ-C du ing e hylene-p omo ed abscission. The AZ-C consis s o wo di e en cell a eas, he Di ided Cells A ea (DCA) and he
S a ch- ich A ea (SA). The ea ly cellula and molecula e en s associa ed wi h ci us ui abscission occu in he cen al co e o he AZ-C be ween he axial ascula
bundles and sp ead up o he calyx bu on pe iphe y eaching hen he lo al disc. The inal ou come o his cell sepa a ion p ocess is he shedding o he ui
emaining he calyx bu on a ached o he ee as shown in he inse o he uppe -le co ne o he igu e. Two pa allel cellula e en s in ol ing cell wall dissolu ion and
syn hesis and deposi ion o lignin occu ed speci ically in he SA o he AZ-C cells du ing abscission. These cellula e en s a e po en ially p omo ed by he
issue-speci ic exp ession o pa icula membe s o se e al gene amilies ha ha e been clea ly in ol ed in hose me abolic pa hways.
Changes in he pec ic polysaccha ides dis ibu ion in he
AZ-C cell walls enabled us o co ela e e idences o enzyma ic
ac i i y wi h gene exp ession esul s. In pa icula , based on
immunode ec ion o pa ially me hyles e i ied/de-es e i ied HG
and exp ession esul s, we p opose ha PMEs Ci PME24 and
Ci PME41 and PAE Ci PAE4 may ac on de-es e i ica ion o
HGs in he AZ-C cell walls (Table 1,Figu e S3). The ac i i y o
PMEs and PAEs is hough o acili a e he subsequen ac ion
o pec in hyd olases (Chen and Ma , 1996). Thus, he PGs
Ci PG43,Ci PG16,Ci PG20,Ci PG41, and Ci PG42, and he PLs
Ci PL5, and Ci PL19 may po en ially hyd olyze he HGs highly
accessible a e Ci PME24,Ci PME41, and Ci PAE4 ac i i y
(Table 1,Figu es 5,6,Figu es S3–S5). Finally, he only α-L-
a abino u anosidase iden i ied in ci us (Ci ASD1) did no show
signi ican changes in gene exp ession based on he s a is ical
cu o men ioned in ma e ials and me hods (Figu e S4). In
A abidopsis, i has been epo ed ha A BXL1 and A BXL3 ac ed
as bi unc ional α-L-a abino u anosidase/β-D-xylosidases (Minic
e al., 2004; A so ski e al., 2009). The e o e, changes obse ed in
(1, 5)-α-L-a abinans a he AZ-C migh be due o he dual ac i i y
o β-XYLs such as Ci BXL11 and Ci BXL19, which we e in he
same clade as A BXL1 and A BXL3 (Figu e S4).
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Me elo e al. Cell Wall Remodeling du ing Abscission
A Se o Genes In ol ed in Lignin
Biosyn hesis and Polyme iza ion a e
Regula ed in he AZ-C Cells
Signi ican exp essed genes belonging o di e en gene amilies
in ol ed he monolignol biosyn hesis pa hway we e up-
egula ed by e hylene exclusi ely in he AZ-C (Table 2,
Figu e 8,Figu e S7). These included a phenylalanine ammonia-
lyase (Ci PAL5), a p-couma a e 3-hyd oxylase (Ci C3H1), a
hyd oxycinnamoyl-CoA shikima e/quina e hyd oxycinnamoyl
ans e ase (Ci HCT2), a 4-couma a e-CoA ligase-like p o ein
(Ci 4CL7), a cinnamoyl-CoA educ ase-like p o ein (Ci CCR1),
and a cinnamyl alcohol dehyd ogenase (Ci CAD3). In addi ion,
h ee genes encoding p o eins in ol ed in he oxida i e coupling
o monolignols and belonging o he CASPARIAN STRIP
MEMBRANE DOMAIN PROTEIN amily (Ci CASPL1D1a,
Ci CASPL2B2, and Ci CASPL4A4) we e also up- egula ed
by e hylene exclusi ely in he AZ-C (Table 2,Figu e 8,
Figu e S7). These esul s co ela ed wi h he obse a ion o lignin
deposi ion in he AZ-C a e 24 and 48 h o e hylene/ACC
ea men (Figu es 1B,C) and he inc ease in he le el o
lignin in e media es de ec ed by UPLC-MS/MS in AZ-C cells
(Figu e 8B). A signi ican inc ease o couma ic acid was obse ed
a 12 h a e ACC ea men . In addi ion, le els o ca eic acid and
e ulic acid, compounds ha a e syn hesized om couma ic acid,
inc eased a 12 h and we e main ained up o 36 h. Taken oge he ,
hese da a mainly e lec ed he ac i a ion o he H lignin pa hway
which esul s om he inco po a ion o p-hyd oxyphenyl (H)
uni s in o he lignin polyme (Figu e 8C). Howe e , G lignin
(lignin wi h guaiacyl uni s) biosyn hesis would be also possible in
ac i a ed AZ-C cells since an inc ease in Ci C3H1 and Ci HCT2
exp ession and ca eic and e ulic acids le els also occu ed in
AZ-C cells despi e any membe o he CCoAOMTs gene amily
we e up- egula ed (Figu e 8). Rega ding S lignin biosyn hesis,
ou ca eic acid O-me hyl ans e ases (Ci COMT2,Ci COMT3,
Ci COMT6, and Ci COMT12) we e down- egula ed exclusi ely
in AZ-C cells (Table 2,Figu e 8C). Ci COMT3 was closely
ela ed in sequence o A COMT1 (Figu e S7), a membe o he
A abidopsis COMT gene amily wi h 5-hyd oxyconi e aldehyde
O-me hyl ans e ase ac i i y ha has been implica ed di ec ly
in S lignin syn hesis (Naka subo e al., 2008). These esul s
sugges ha he S lignin pa hway migh be inac i e in AZ-C
cells du ing ui abscission. In woody dico yledons, such as
ci us, lignin is polyme ized om mos ly G and S lignin subuni s
(Sa kanen and Ludwig, 1971). Howe e , lignin composi ion can
di e among cell ypes (Nakashima e al., 2008; Ruel e al., 2009)
and exp ession da a sugges ed ha cell walls o AZ-C cells migh
be mainly en iched in H lignin and p obably also in G lignin
subuni s.
The ole o lignin deposi ion has been associa ed wi h
he gene a ion o p o ec i e laye s a he issues emaining
in he plan du ing he las s ep o he abscission p ocess
(Addico , 1982; Agus í e al., 2008; Van Nocke , 2009).
Howe e , i has been sugges ed ha ligni ica ion could also
acili a e he mechanical cell wall b eakage du ing cell sepa a ion
p ocesses (Sex on, 1979; Liljeg en e al., 2000). In he AZ-
C, lignin deposi ion only occu ed a he dis al side o
he AZ-C (Figu e 8A). This di e en ial deposi ion o lignin
s ongly sugges s ha his polyme mainly ac s by gene a ing
a ension in he ac u e plane o acili a e cell wall b eakage
du ing ci us ui abscission a he han o ming p o ec i e
laye s.
CONCLUSION
In his wo k, he isola ion o specialized cell ypes h ough LM,
combined wi h he global ansc ip ional analysis o e hylene-
p omo ed AZ-C cells and he compa ison wi h adjacen FR cells,
enabled us o iden i y an AZ-C-exclusi e gene se po en ially
in ol ed in ci us ui abscission. This se o genes includes hose
ela ed o cell wall emodeling as well as lignin biosyn hesis and
polyme iza ion. The combined unc ion o hese genes would
enable cell wall modi ica ions necessa y o o gan de achmen
(Figu e 9). These esul s, oge he wi h he ana omical and
mo phological analysis o he AZ-C, he de e mina ion o
changes in pec ic polysaccha ides dis ibu ion and he deposi ion
o ex acellula polyme s obse ed in he ac i a ed AZ-C, lead
o he mos comp ehensi e cha ac e iza ion o ci us ui
abscission pe o med o da e. In addi ion, ou wo k shows he
i s classi ica ion in ci us o gene amilies in ol ed in cell
wall modi ica ion, which a e c ucial in abscission, and e eals
a obus nexus be ween phylogene ic p oximi y and exp ession
pa e n du ing abscission in ci us and o he plan species, no
p e iously desc ibed. The e o e, his s udy s ongly sugges s
ha di e en plan species use common genes o con ol he
abscission p ocess. The da ase p o ided in his s udy is a highly
aluable esou ce o guiding u u e unc ional analyses in o de
o answe speci ic abscission- ela ed ques ions. In pa icula ,
hose cell wall- ela ed genes, which a e e olu iona ily conse ed
in ci us and o he plan species wi h simila exp ession pa e n
du ing abscission, would ep esen majo candida e genes o
u he bio echnological app oaches.
AUTHOR CONTRIBUTIONS
PM, JA, MT, and FT concei ed he su ey and designed he
expe imen s; PM pe o med lase mic odissec ion o ci us
issues; VA and AG pe o med me aboli e p o iling analyses; PM,
JA, and CD pe o med mic oa ay expe imen s and analyses;
MC, SC, and FT pe o med immunolocaliza ion expe imen s;
LE, MG, MP, and FT pe o med in si u hyb idiza ion
expe imen s; PM, JA, MT, and FT w o e he a icle wi h
con ibu ions o all he au ho s; all au ho s discussed he esul s
and edi ed he a icle.
ACKNOWLEDGMENTS
We hank E. Blázquez, I. Sanchís, A. Boix, and M. A. A gomániz
o he help on all o he assays and expe imen s pe o med bo h
in he labo a o y and in he ield. This s udy was inancially
suppo ed by he Spanish Ins i u o Nacional de In es igaciones
Ag a ias (g an RTA2008-00065-00-00 o FT [including a PhD
ellowship o PM] and RTA2014-00071-C06-01 o MT), he
F on ie s in Plan Science | www. on ie sin.o g 17 Feb ua y 2017 | Volume 8 | A icle 126
Me elo e al. Cell Wall Remodeling du ing Abscission
Spanish Minis e io de Economia e Inno ación (g an s PSE-
060000-2009-8 and IPT-010000-2010-43 o MT and BIO2011-
26302 o MP) and he Spanish Minis e io de Indus ia (g an
AGL2011-30240 o MT). VA and CD we e ecipien s o a
“Juan de la Cie a” and an INIA/CCAA pos doc o al con ac ,
espec i ely.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound
online a : h p://jou nal. on ie sin.o g/a icle/10.3389/ pls.2017.
00126/ ull#supplemen a y-ma e ial
Table S1 | Speci ic p ime s used o sqRT-PCR.
Table S2 | Genes egula ed by e hylene exclusi ely in AZ-C o FR cells,
and in bo h AZ-C and FR cells.
Table S3 | Func ional ca ego ies ela ed o polysaccha ide me abolism
and subcellula localiza ion egula ed in he AZ-C and he FR.
Table S4 | Iden i ica ion o ci us genes belonging o di e en amilies o
cell wall emodeling enzymes, and monolignol biosyn hesis and
polyme iza ion.
Figu e S1 | sqRT-PCR-based ela i e exp ession in he AZ-C and he
FR.
Figu e S2 | LM isola ion o AZ-C and FR cells.
Figu e S3 | Phylogene ic ela ionships be ween Ca bohyd a e es e ases.
Figu e S4 | Phylogene ic ela ionships be ween Glycoside hyd olases.
Figu e S5 | Phylogene ic ela ionships be ween Pec a e lyases.
Figu e S6 | Phylogene ic ela ionships be ween Expansins.
Figu e S7 | Phylogene ic ela ionships be ween membe s o gene amilies
associa ed wi h monolignol biosyn hesis and polyme iza ion.
REFERENCES
Addico , F. T. (1982). Abscission. Uni e si y o Cali o nia P ess, Be keley.
Agus í, J., Gimeno, J., Me elo, P., Se ano, R., Ce cos, M., Conesa, A., e al. (2012).
Ea ly gene exp ession e en s in he lamina abscission zone o abscission-
p omo ed ci us lea es a e a cycle o wa e s ess/ ehyd a ion: in ol emen
o Ci bHLH1. J. Exp. Bo . 63, 6079–6091. doi: 10.1093/jxb/e s270
Agus í, J., Me elo, P., Ce cos, M., Tadeo, F. R., and Talon, M. (2008). E hylene-
induced di e en ial gene exp ession du ing abscission o ci us lea es. J. Exp.
Bo . 59, 2717–2733. doi: 10.1093/jxb/e n138
Agus í, J., Me elo, P., Ce cos, M., Tadeo, F. R., and Talon, M. (2009).
Compa a i e ansc ip ional su ey be ween lase -mic odissec ed cells om
lamina abscission zone and pe iola co ical issue du ing e hylene-p omo ed
abscission in ci us lea es. BMC Plan Biol. 9:127. doi: 10.1186/1471-2229-9-127
A gamasilla, R., Gómez-Cadenas, A., and A bona, V. (2014). Me abolic and
egula o y esponses in ci us oo s ocks in esponse o ad e se en i onmen al
condi ions. J. Plan G ow h Regul. 33, 169–180. doi: 10.1007/s00344-013-
9359-z
A so ski, A. A., Popma, T. M., Haughn, G. W., Ca pi a, N. C., McCann,
M. C., and Wes e n, T. L. (2009). A BXL1 encodes a bi unc ional
be a-D-xylosidase/alpha-L-a abino u anosidase equi ed o pec ic a abinan
modi ica ion in A abidopsis mucilage sec e o y cells. Plan Physiol. 150,
1219–1234. doi: 10.1104/pp.109.138388
A kinson, R. G., Sch ode , R., Halle , I. C., Cohen, D., and Mac ae, E. A. (2002).
O e exp ession o polygalac u onase in ansgenic apple ees leads o a ange
o no el pheno ypes in ol ing changes in cell adhesion. Plan Physiol. 129,
122–133. doi: 10.1104/pp.010986
B ummell, D. A., Hall, B. D., and Benne , A. B. (1999). An isense supp ession o
oma o endo-1,4-be a-glucanase Cel2 mRNA accumula ion inc eases he o ce
equi ed o b eak ui abscission zones bu does no a ec ui so ening. Plan
Mol. Biol. 40, 615–622. doi: 10.1023/A:1006269031452
Bu ns, J. K. (2002). Using molecula biology ools o iden i y abscission ma e ials
o ci us. Ho Science 37, 459–464.
Bu ns, J. K., Lewandowski, D. J., Nai n, C. J., and B own, G. E. (1998).
Endo-1,4-β-glucanase gene exp ession and cell wall hyd olase ac i i ies
du ing abscission in Valencia o ange. Physiol. Plan . 102, 217–225.
doi: 10.1034/j.1399-3054.1998.1020209.x
Can a el, B. L., Cou inho, P. M., Rancu el, C., Be na d, T., Lomba d, V., and
Hen issa , B. (2009). The Ca bohyd a e-Ac i e EnZymes da abase (CAZy):
an expe esou ce o glycogenomics. Nucleic Acids Res. 37, D233–D238.
doi: 10.1093/na /gkn663
Ca uso, M., Me elo, P., Dis e ano, G., La Mal a, S., Lo Pie o, A. R., Tadeo, F. R.,
e al. (2012). Compa a i e ansc ip ome analysis o s yla canal cells iden i ies
no el candida e genes implica ed in he sel -incompa ibili y esponse o Ci us
clemen ina.BMC Plan Biol. 12:20. doi: 10.1186/1471-2229-12-20
Ce cos, M., Sole , G., Iglesias, D. J., Gadea, J., Fo men , J., and Talon, M. (2006).
Global analysis o gene exp ession du ing de elopmen and ipening o ci us
ui lesh. A p oposed mechanism o ci ic Acid u iliza ion. Plan Mol. Biol.
62, 513–527. doi: 10.1007/s11103-006-9037-7
Chen, E. M. W., and Ma , A. J. (1996). Na u e o si es hyd olyzable by
endopolygalac u onase in pa ially-es e i ied homogalac u onans. Ca bohyd .
Polym. 29, 129–136. doi: 10.1016/0144-8617(96)00005-7
Cheng, C., Zhang, L., Yang, X., and Zhong, G. (2015). P o iling gene exp ession
in ci us ui calyx abscission zone (AZ-C) ea ed wi h e hylene. Mol. Gene .
Genomics 290, 1991–2006. doi: 10.1007/s00438-015-1054-2
Coimb a, S., Almeida, J., Junquei a, V., Cos a, M. L., and Pe ei a, L. G. (2007).
A abinogalac an p o eins as molecula ma ke s in A abidopsis haliana sexual
ep oduc ion. J. Exp. Bo . 58, 4027–4035. doi: 10.1093/jxb/e m259
Co bacho, J., Romoja o, F., Pech, J. C., La che, A., and Gomez-Jimenez,
M. C. (2013). T ansc ip omic e en s in ol ed in melon ma u e- ui
abscission comp ise he sequen ial induc ion o cell-wall deg ading genes
coupled o a s imula ion o endo and exocy osis. PLoS ONE 8:e58363.
doi: 10.1371/jou nal.pone.0058363
Es o nell, L. H., Agus i, J., Me elo, P., Talon, M., and Tadeo, F. R. (2013).
Elucida ing mechanisms unde lying o gan abscission. Plan Sci. 199–200,
48–60. doi: 10.1016/j.plan sci.2012.10.008
Fe a ese, L., T aino i, L., Mo e o, P., Pol e ino De Lau e o, P., Rascio, N., and
Casado o, G. (1995). Di e en ial e hylene-inducible exp ession o cellulase in
peppe plan s. Plan Mol. Biol. 29, 735–747. doi: 10.1007/BF00041164
Gallasch, P. (1996). “E alua ing new selec ions o la e hanging na el o ange,” in
In e na ional Ci us Cong ess (Sun Ci y: In e na ional Socie y o Ci icul u e).
Gil-Amado, J. A., and Gomez-Jimenez, M.C. (2013). T ansc ip ome analysis
o ma u e ui abscission con ol in oli e. Plan Cell Physiol. 54, 244–269.
doi: 10.1093/pcp/pcs179
Gomez, M. D., U bez, C., Pe ez-Amado , M. A., and Ca bonell, J. (2011).
Cha ac e iza ion o cons ic ed ui (c ) mu an unco e s a ole o
A MYB117/LOF1 in o ule and ui de elopmen in A abidopsis haliana.PLoS
ONE 6:e18760. doi: 10.1371/jou nal.pone.0018760
Gonzalez-Ca anza, Z. H., Ellio , K. A., and Robe s, J. A. (2007). Exp ession
o polygalac u onases and e idence o suppo hei ole du ing cell
sepa a ion p ocesses in A abidopsis haliana.J. Exp. Bo . 58, 3719–3730.
doi: 10.1093/jxb/e m222
Gonzalez-Ca anza, Z. H., Whi elaw, C. A., Swa up, R., and Robe s, J. A.
(2002). Tempo al and spa ial exp ession o a polygalac u onase du ing lea and
lowe abscission in oilseed ape and A abidopsis. Plan Physiol. 128, 534–543.
doi: 10.1104/pp.010610
Go en, R. (1993). Ana omical, physiological, and ho monal aspec s o abscission
in ci us. Ho . Re . 15:182. doi: 10.1002/9780470650547.ch4
Had ield, K. A., Rose, J. K. C., Ya e , D. S., Be ka, R. M., and Benne , A. B.
(1998). Polygalac u onase gene exp ession in ipe melon ui suppo s a ole
o polygalac u onase in ipening-associa ed pec in disassembly. Plan Physiol.
117, 363–373. doi: 10.1104/pp.117.2.363
Hen issa , B. (1991). A classi ica ion o glycosyl hyd olases based on amino acid
sequence simila i ies. Biochem. J. 280, 309–316. doi: 10.1042/bj2800309
F on ie s in Plan Science | www. on ie sin.o g 18 Feb ua y 2017 | Volume 8 | A icle 126
Me elo e al. Cell Wall Remodeling du ing Abscission
Hong, S. B., and Tucke , M. L. (1998). Genomic o ganiza ion o six oma o
polygalac u onases and 5′ups eam sequence iden i y wi h ap1 and win2
genes. Mol. Gen. Gene . 258, 479–487. doi: 10.1007/s004380050758
Hube man, M., Go en, R., and Zamski, E. (1983). Ana omical aspec s
o ho monal egula ion o abscission in ci us – The shoo -peduncle
abscission zone in he non-abscising s age. Physiol. Plan . 59, 445–454.
doi: 10.1111/j.1399-3054.1983. b04228.x
Jones, L., Seymou , G. B., and Knox, J. P. (1997). Localiza ion o pec ic galac an in
oma o cell walls using a monoclonal an ibody speci ic o (1[−>]4)-[be a]-D-
galac an. Plan Physiol. 113, 1405–1412. doi: 10.1104/pp.113.4.1405
Kalai zis, P., Koehle , S. M., and Tucke , M. L. (1995). Cloning o a oma o
polygalac u onase exp essed in abscission. Plan Mol. Biol. 28, 647–656.
doi: 10.1007/BF00021190
Kalai zis, P., Solomos, T., Tucke , M. L. (1997). Th ee di e en polygalac u onases
a e exp essed in oma o lea and lowe abscission, each wi h a
di e en empo al exp ession pa e n. Plan Physiol. 113, 1303–1308.
doi: 10.1104/pp.113.4.1303
Kim, J., Shiu, S.-H., Thoma, S., Li, W.-H., and Pa e son, S. E. (2006). Pa e ns
o expansion and exp ession di e gence in he plan polygalac u onase gene
amily. Genome Biol. 7:R87. doi: 10.1186/gb-2006-7-9- 87
Knox, J. P., Lins ead, P. J., King, J., Coope , C., and Robe s, K. (1990).
Pec in es e i ica ion is spa ially egula ed bo h wi hin cell walls and
be ween de eloping issues o oo apices. Plan a 181, 512–521.
doi: 10.1007/BF00193004
Konishi, S., Izawa, T., Lin, S. Y., Ebana, K., Fuku a, Y., Sasaki, T., e al. (2006).
An SNP caused loss o seed sha e ing du ing ice domes ica ion. Science 312,
1392–1396. doi: 10.1126/science.1126410
Kuma , S., S eche , G., and Tamu a, K. (2016). MEGA7: Molecula E olu iona y
Gene ics Analysis e sion 7.0 o bigge da ase s. Mol. Biol. E ol. 33, 1870–1874.
doi: 10.1093/molbe /msw054
Lashb ook, C. C., and Cai, S. (2008). Cell wall emodeling in A abidopsis s amen
abscission zones: empo al aspec s o con ol in e ed om ansc ip ional
p o iling. Plan Signal. Beha . 3, 733–736. doi: 10.4161/psb.3.9.6489
Le unic, I., Doe ks, T., and Bo k, P. (2009). SMART 6: ecen upda es and new
de elopmen s. Nucleic Acids Res. 37, D229–D232. doi: 10.1093/na /gkn808
Li, P., Lee, S.-H., and Hsu, H.-Y. (2011). Re iew on ui ha es ing me hod o
po en ial use o au oma ic ui ha es ing sys ems. P oc. Eng. 23, 351–366.
doi: 10.1016/j.p oeng.2011.11.2514
Li, C., Wang, Y., Ying, P., Ma, W., and Li, J. (2015). Genome-wide digi al ansc ip
analysis o pu a i e ui le abscission ela ed genes egula ed by e hephon in
li chi. F on . Plan Sci. 6:502. doi: 10.3389/ pls.2015.00502
Li, J. G., and Yuan, R. C. (2008). NAA and e hylene egula e exp ession o genes
ela ed o e hylene biosyn hesis, pe cep ion, and cell wall deg ada ion du ing
ui abscission and ipening in ‘Delicious’ apples. J. Plan G ow h. Regul. 27,
283–295. doi: 10.1007/s00344-008-9055-6
Libe ini, E., Li, Y., and McQueen-Mason, S. J. (2004). Phylogene ic analysis
o he plan endo-be a-1,4-glucanase gene amily. J. Mol. E ol. 58, 506–515.
doi: 10.1007/s00239-003-2571-x
Liljeg en, S. J., Di a, G. S., Eshed, Y., Sa idge, B., Bowman, J. L., and Yano sky,
M. F. (2000). SHATTERPROOF MADS-box genes con ol seed dispe sal in
A abidopsis. Na u e 404, 766–770. doi: 10.1038/35008089
Liu, B., Bu enko, M. A., Shi, C. L., Boli a , J. L., Winge, P., S en ik, G. E., e al.
(2013). NEVERSHED and INFLORESCENCE DEFICIENT IN ABSCISSION
a e di e en ially equi ed o cell expansion and cell sepa a ion du ing
lo al o gan abscission in A abidopsis haliana.J. Exp. Bo . 64, 5345–5357.
doi: 10.1093/jxb/e 232
Lomba d, V., Golaconda Ramulu, H., D ula, E., Cou inho, P. M., and Hen issa ,
B. (2014). The ca bohyd a e-ac i e enzymes da abase (CAZy) in 2013. Nucleic
Acids Res. 42, D490–D495. doi: 10.1093/na /gk 1178
Ma inez-Godoy, M. A., Mau i, N., Jua ez, J., Ma ques, M. C., San iago, J.,
Fo men , J., e al. (2008). A genome-wide 20 K ci us mic oa ay o gene
exp ession analysis. BMC Genomics 9:318. doi: 10.1186/1471-2164-9-318
Ma as, A. J., Agus í, J., Tadeo, F. R., Talón, M., and Rose, J. K. C. (2010).
Tissue-speci ic ansc ip ome p o iling o he ci us ui epide mis and
subepide mis using lase cap u e mic odissec ion. J. Exp. Bo . 61, 3321–3330.
doi: 10.1093/jxb/e q153
Mbéguié-a-Mbéguié, D., Hube , O., Bau ens, F. C., Ma sumo o, T., Chille , M.,
Fils-Lycaon, B., e al. (2009). Exp ession pa e ns o cell wall-modi ying genes
om banana du ing ui ipening and in ela ionship wi h inge d op. J. Exp.
Bo . 60, 2021–2034. doi: 10.1093/jxb/e p079
Mei , S., Philosoph-Hadas, S., Sunda esan, S., Sel a aj, K. S. V., Bu d, S., Ophi ,
R., e al. (2010). Mic oa ay analysis o he abscission- ela ed ansc ip ome in
he oma o lowe abscission zone in esponse o auxin deple ion. Plan Physiol.
154, 1929–1956. doi: 10.1104/pp.110.160697
Minic, Z., Rihouey, C., Do, C. T., Le ouge, P., and Jouanin, L. (2004). Pu i ica ion
and cha ac e iza ion o enzymes exhibi ing β-D-xylosidase ac i i ies in s em
issues o a abidopsis. Plan Physiol. 135, 867–878. doi: 10.1104/pp.104.041269
Nakashima, J., Chen, F., Jackson, L., Shadle, G., and Dixon, R. A. (2008). Mul i-si e
gene ic modi ica ion o monolignol biosyn hesis in al al a (Medicago sa i a):
e ec s on lignin composi ion in speci ic cell ypes. New Phy ol. 179, 738–750.
doi: 10.1111/j.1469-8137.2008.02502.x
Naka subo, T., Ki amu a, Y., Sakakiba a, N., Mizu ani, M., Ha o i, T.,
Saku ai, N., e al. (2008). A 5g54160 gene encodes A abidopsis haliana
5-hyd oxyconi e aldehyde O-me hyl ans e ase. J. Wood Sci. 54, 312–317.
doi: 10.1007/s10086-008-0958-4
Niede hu h, C. E., Pa ha ka , O. R., and Walke , J. C. (2013). T ansc ip ional
p o iling o he A abidopsis abscission mu an hae hsl2 by RNA-Seq. BMC
Genomics 14:37. doi: 10.1186/1471-2164-14-37
Ogawa, M., Kay, P., Wilson, S., and Swain, S. M. (2009). ARABIDOPSIS
DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2,
and QUARTET2 a e Polygalac u onases equi ed o cell sepa a ion
du ing ep oduc i e de elopmen in A abidopsis. Plan Cell 21, 216–233.
doi: 10.1105/ pc.108.063768
Pa a, R., Pa edes, M., Sanchez-Calle, I., and Gomez-Jimenez, M. C.
(2013). Compa a i e ansc ip ional p o iling analysis o oli e ipe- ui
pe ica p and abscission zone issues shows exp ession di e ences and
dis inc pa e ns o ansc ip ional egula ion. BMC Genomics 14:866.
doi: 10.1186/1471-2164-14-866
Peng, G., Wu, J., Lu, W., and Li, J. (2013). A polygalac u onase gene clus e ed
in o clade E in ol ed in lychee ui le abscission. Sci. Ho ic. 150, 244–250.
doi: 10.1016/j.scien a.2012.10.029
Pe e sen, M., Sande , L., Child, R., Van Onckelen, H., Ul sko , P., and
Bo kha d , B. (1996). Isola ion and cha ac e isa ion o a pod dehiscence zone-
speci ic polygalac u onase om B assica napus.Plan Mol. Biol. 31, 517–527.
doi: 10.1007/BF00042225
Picke sgill, B. (2007). Domes ica ion o plan s in he Ame icas: insigh s
om mendelian and molecula gene ics. Ann. Bo . 100, 925–940.
doi: 10.1093/aob/mcm193
Robe s, J. A., Ellio , K. A., and Gonzalez-Ca anza, Z. H. (2002). Abscission,
dehiscence, and o he cell sepa a ion p ocesses. Annu. Re . Plan Biol. 53,
131–158. doi: 10.1146/annu e .a plan .53.092701.180236
Roongsa ham, P., Mo cillo, F., Jan asu iya a , C., Pizo , M., Moussu, S., Jayawee a,
D., e al. (2012). Tempo al and spa ial exp ession o polygalac u onase gene
amily membe s e eals di e gen egula ion du ing leshy ui ipening
and abscission in he monoco species oil palm. BMC Plan Biol. 12:150.
doi: 10.1186/1471-2229-12-150
Ruel, K., Be io-Sie a, J., De ik and, M. M., Polle , B., The enin, J.,
Lapie e, C., e al. (2009). Impac o CCR1 silencing on he assembly o
ligni ied seconda y walls in A abidopsis haliana.New Phy ol. 184, 99–113.
doi: 10.1111/j.1469-8137.2009.02951.x
Sande , L., Child, R., Ul sko , P., Alb ech sen, M., and Bo kha d , B. (2001).
Analysis o a dehiscence zone endo-polygalac u onase in oilseed ape (B assica
napus) and A abidopsis haliana: e idence o oles in cell sepa a ion in
dehiscence and abscission zones, and in s yla issues du ing pollen ube
g ow h. Plan Mol. Biol. 46, 469–479. doi: 10.1023/A:1010619002833
Sa kanen, K. V., and Ludwig, C. H. (1971). Lignins: Occu ence, Fo ma ion,
S uc u e and Reac ions. New Yo k, NY: Wiley-In e science.
Schul z, J., Milpe z, F., Bo k, P., and Pon ing, C. P. (1998). SMART, a simple
modula a chi ec u e esea ch ool: iden i ica ion o signaling domains. P oc.
Na l. Acad. Sci. U.S.A. 95, 5857–5864. doi: 10.1073/pnas.95.11.5857
Sex on, R. (1979). Spa ial and empo al aspec s o cell sepa a ion in he
olia abscission zones o Impa iens sul ani Hook. P o oplasma 99, 53–66.
doi: 10.1007/BF01274069
Shi aishi, M., and Yanagisawa, T. (1988). Ana omical and his ochemical s udies o
s a ch dis ibu ion du ing abscission in ilamen s and pe als o Wase Sa suma
manda in. J. Jpn. Soc. Ho ic. Sci. 56, 365–374. doi: 10.2503/jjshs.56.365
F on ie s in Plan Science | www. on ie sin.o g 19 Feb ua y 2017 | Volume 8 | A icle 126
Me elo e al. Cell Wall Remodeling du ing Abscission
Singh, A. P., Pandey, S. P., Rajluxmi, Pandey, S., Na h, P., and Sane,
A. P. (2011). T ansc ip ional ac i a ion o a pec a e lyase gene, RbPel1,
du ing pe al abscission in ose. Pos ha es Biol. Technol. 60, 143–148.
doi: 10.1016/j.pos ha bio.2010.12.014
Spiegel-Roy, P., and Goldschmid , E. E. (1996). The Biology o Ci us. Camb idge:
Camb idge Uni e si y P ess.
Sun, L., and Van Nocke , S. (2010). Analysis o p omo e ac i i y o membe s o
he PECTATE LYASE-LIKE (PLL) gene amily in cell sepa a ion in A abidopsis.
BMC Plan Biol. 10:152. doi: 10.1186/1471-2229-10-152
Tadeo, F. R., and P imo-Millo, E. (1990). Pe oxidase ac i i y changes and lignin
deposi ion du ing he senescence p ocess in ci us s igmas and s yles. Plan Sci.
68, 47–56. doi: 10.1016/0168-9452(90)90151-D
Tonu i, P., Cass, L. G., and Ch is o e sen, R. E. (1995). The exp ession o cellulase
gene amily membe s du ing induced a ocado ui abscission and ipening.
Plan Cell En i on. 18, 709–713. doi: 10.1111/j.1365-3040.1995. b00573.x
Tucke M. L., and Kim, J. (2015). Abscission esea ch: wha we know and wha we
s ill need o s udy. S ewa Pos ha es Re . 2:1. doi: 10.2212/sp .2015.2.1
Tucke , M. L., Bu ke, A., Mu phy, C. A., Thai, V. K., and Eh en ied, M. L.
(2007). Gene exp ession p o iles o cell wall-modi ying p o eins associa ed
wi h soybean cys nema ode in ec ion, pe iole abscission, oo ips, lowe s,
apical buds, and lea es. J. Exp. Bo . 58, 3395–3406. doi: 10.1093/jxb/e m188
U banowicz, B. R., Benne , A. B., Del Campillo, E., Ca ala, C., Hayashi,
T., Hen issa , B., e al. (2007). S uc u al o ganiza ion and a s anda dized
nomencla u e o plan endo-1,4-be a-glucanases (cellulases) o glycosyl
hyd olase amily 9. Plan Physiol. 144, 1693–1696. doi: 10.1104/pp.107.102574
Van Nocke , S. (2009). De elopmen o he abscission zone. S ewa Pos ha es
Re . 5, 1–6. doi: 10.2212/sp .2009.1.5
Willa s, W. G. T., Ma cus, S. E., and Knox, J. P. (1998). Gene a ion o a
monoclonal an ibody speci ic o (1→5)-α-l-a abinan. Ca bohyd . Res. 308,
149–152. doi: 10.1016/S0008-6215(98)00070-6
Wilson, W. C., and Hende sho , C. (1968). Ana omical and his ochemical s udies
o abscission o o anges. P oc. Am. Soc. Ho ic. Sci. 92, 203–210.
Wu, G. A., P ochnik, S., Jenkins, J., Salse, J., Hells en, U., Mu a , F., e al.
(2014). Sequencing o di e se manda in, pummelo and o ange genomes e eals
complex his o y o admix u e du ing ci us domes ica ion. Na . Bio echnol. 32,
656–662. doi: 10.1038/nb .2906
Wu, X.-M., Yu, Y., Han, L.-B., Li, C.-L., Wang, H.-Y., Zhong, N.-Q.,
e al. (2012). The obacco BLADE-ON-PETIOLE2 gene media es
di e en ia ion o he co olla abscission zone by con olling longi udinal
cell expansion. Plan Physiol. 159, 835–850. doi: 10.1104/pp.112.
193482
Zaca ias, L., Tadeo, F. R., Bono, R., and P imo-Millo, E. (1993). “Abscission s udies
in a new mu an o na el o anges,” in Cellula and Molecula Aspec s o he Plan
Ho mone E hylene: P oceedings o he In e na ional Symposium on Cellula and
Molecula Aspec s o Biosyn hesis and Ac ion o he Plan Ho mone E hylene,
Agen, F ance, Augus 31–Sep embe 4, 1992, eds J. C. Pech, A. La ché, and C.
Balagué (Do d ech : Sp inge ), 284–290.
Zhu, H., Da dick, C., Bee s, E., Callanhan, A., Xia, R., and Yuan, R. (2011).
T ansc ip omics o shading-induced and NAA-induced abscission in apple
(Malus domes ica) e eals a sha ed pa hway in ol ing educed pho osyn hesis,
al e a ions in ca bohyd a e anspo and signaling and ho mone c oss alk.
BMC Plan Biol. 11:138. doi: 10.1186/1471-2229-11-138
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