RESEARCH ARTICLE
AINTEGUMENTA and he D- ype cyclin CYCD3;1 egula e oo
seconda y g ow h and espond o cy okinins
Rica do S. Randall
1,‡
, Shunsuke Miyashima
2,‡
, Tiina Bloms e
3
, Jing Zhang
3
, Annakaisa Elo
3
, Anna Ka lbe g
4
,
Juha Immanen
3
, Kaisa Nieminen
3
, Ji-Young Lee
5
, Ta suo Kakimo o
2
, Ka olina Blajecka
6
, Cha les W. Melnyk
6
,
Anne e Alcasabas
1,
*, Celine Fo zani
1
, Miho Ma sumo o-Ki ano
2
, A i Pekka Ma
ho
nen
3
, Rishikesh Bhale ao
4
,
Wal e Dewi e
1
,Yka
Hela iu a
6,§
and James A. H. Mu ay
1,§
ABSTRACT
Highe plan ascula u e is cha ac e ized by wo dis inc
de elopmen al phases. Ini ially, a well-de ined adial p ima y pa e n
is es ablished. In eudico s, his is ollowed by seconda y g ow h,
which in ol es de elopmen o he cambium and is equi ed o
e icien wa e and nu ien anspo and wood o ma ion. Regula ion
o seconda y g ow h in ol es se e al phy oho mones, and cy okinins
ha e been implica ed as key playe s, pa icula ly in he ac i a ion o
cell p oli e a ion, bu he molecula mechanisms media ing his
ho monal con ol emain unknown. He e we show ha he genes
encoding he ansc ip ion ac o AINTEGUMENTA (ANT) and he
D- ype cyclin CYCD3;1 a e exp essed in he ascula cambium o
A abidopsis oo s, espond o cy okinins and a e bo h equi ed o
p ope oo seconda y hickening. Cy okinin egula ion o ANT and
CYCD3 also occu s du ing seconda y hickening o popla s ems,
sugges ing his ep esen s a conse ed egula o y mechanism.
KEY WORDS: Cy okinins, Seconda y g ow h, Cyclin D,
AINTEGUMENTA, Roo de elopmen
INTRODUCTION
The phy oho mone cy okinin egula es se e al oo de elopmen al
p ocesses, including elonga ion, apical me is em main enance and
ascula mo phogenesis (Fe ei a and Kiebe , 2005; Higuchi e al.,
2004; Mahonen e al., 2000; Nishimu a e al., 2004). Cy okinin
signalling, oge he wi h o he ho monal cues (Miyashima e al.,
2013), is also impo an o oo seconda y g ow h, which in ol es
hickening o he oo ia p oli e a ion o cambial cells (Spice and
G oo e , 2010; Nieminen e al., 2004). A abidopsis mu an s
de ec i e in cy okinin biosyn hesis de elop hinne oo s wi h a
cambium composed o ewe cells, a pheno ype escued by
exogenous cy okinin applica ion (Ma sumo o-Ki ano e al., 2008).
Cy okinin he e o e appea s o ac a leas in pa h ough p omo ing
cell di ision and hence ac i a ion o he mi o ic cell cycle.
CYCD genes encode conse ed egula o y sub-uni s o cyclin
D-cyclin-dependen kinase (CYCD-CDK) complexes ha p omo e
cell cycle p og ession in animals and plan s (Menges e al., 2007;
Mo gan, 1997). The CYCD3 subg oup o CYCDs is conse ed
ac oss all highe plan s (Menges e al., 2007) and has h ee membe s
in A abidopsis: CYCD3;1, CYCD3;2 and CYCD3;3. CYCD3s
con ol p og ession h ough he G1/S ansi ion (Menges e al.,
2006), and also egula e he leng h o he empo al pe iod o mi o ic
cell di ision du ing ae ial o gan de elopmen (Dewi e e al., 2007).
CYCD3 genes a e induced by cy okinins (Menges e al., 2007,
2006; Riou-Khamlichi e al., 1999) and a e a e-limi ing o
cy okinin esponses in A abidopsis shoo s (Dewi e e al., 2007;
Riou-Khamlichi e al., 1999). He e, we iden i y no el oles o
CYCD3;1 and he AINTEGUMENTA ansc ip ion ac o in oo
seconda y g ow h. P olonged exp ession o CYCD3;1 in lea es
caused by ec opic exp ession o ANT (Mizukami and Fische , 2000)
has led o he sugges ion ha CYCD3;1 is a a ge o ANT
(Anas asiou and Lenha d, 2007; Wu e al., 2011). Howe e , we
show he e ha CYCD3 and AINTEGUMENTA play independen
oles in egula ing seconda y hickening in oo s, bu p o ide
e idence ha hey a e co- egula ed by cy okinins.
RESULTS AND DISCUSSION
CYCD3;1 is a e-limi ing o oo seconda y hickening
Seconda y g ow h in ol es cell p oli e a ion in he cambium
(Fig. 1A). Gi en he known equi emen o cy okinin signalling o
oo seconda y hickening, and in ol emen o CYCD3s in shoo
g ow h esponses o cy okinins, we analysed he exp ession pa e ns
o CYCD3s using p omo e :GUS cons uc s. pCYCD3;1:GUS
exp ession was obse ed in he inne mos and ou e mos egions
o he s ele o oo s unde going seconda y g ow h (Fig. 1A,B).
These egions con ain he cambium and pe icycle cells espec i ely,
bo h o which con ibu e o seconda y hickening. pCYCD3;2:GUS
and pCYCD3;3:GUS exp ession was de ec ed in he cambium and
in he phloem cells pe pendicula o he p ima y xylem axis.
CYCD3;1 exp ession has also been epo ed in whole moun s o
oo issue unde going seconda y g ow h (Collins e al., 2015) and
he ascula issue o ege a i e and lowe ing A abidopsis shoo
apices (Dewi e e al., 2003). Fu he mo e, exp ession o CYCD3
genes in he ascula issue o oo s unde going p ima y g ow h was
ecen ly in e ed (Collins e al., 2015) om mic oa ay da a ob ained
om luo escen ly ac i a ed cell-so ed p ima y oo cells (B ady
e al., 2007). Collins e al. (2015) also analysed exp ession o
CYCD3 genes in shoo cambium de elopmen wi h he same
ansc ip ional usion epo e lines used he e and de ec ed
exp ession o all h ee CYCD3 genes in his issue. These da a
Recei ed 24 June 2015; Accep ed 5 Augus 2015
1
Depa men o Molecula Biosciences, Ca di School o Biosciences, Ca di
Uni e si y, Ca di , Wales CF10 3AX, UK.
2
Depa men o Biological Sciences,
Osaka Uni e si y, G adua e School o Science, 1-1 Machikaneyama-cho,
Toyonaka, Osaka 560-0043, Japan.
3
Depa men o Biosciences, Ins i u e o
Bio echnology, Viikinkaa i 1 (P.O.Box 65), 00014, Uni e si y o Helsinki, Helsinki,
Finland.
4
Depa men o Plan Physiology, Umeå Uni e si y, Umeå SE-901 87,
Sweden.
5
School o Biological Sciences, College o Na u al Science, Seoul Na ional
Uni e si y, 1 Gwanak- o, Gwanak-gu, Seoul 08826, Ko ea.
6
Sainsbu y Labo a o y,
Camb idge Uni e si y, Ba eman S ee , Camb idge CB2 1LR, UK.
*P esen add ess: BioSyn ha Technology, B oadwa e Road, Welwyn Ga den Ci y
AL7 3AX, UK.
‡
These au ho s con ibu ed equally o his wo k
§
Au ho s o co espondence ([email p o ec ed]; y jo.hela iu a@helsinki. i)
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion
License (h p://c ea i ecommons.o g/licenses/by/3.0), which pe mi s un es ic ed use,
dis ibu ion and ep oduc ion in any medium p o ided ha he o iginal wo k is p ope ly a ibu ed.
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© 2015. Published by The Company o Biologis s L d
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sugges ha CYCD3 genes a e exp essed in adially p oli e a ing
issues and play an ac i e ole in seconda y g ow h.
CYCD3 genes we e ecen ly shown o con ibu e o seconda y
g ow h in A abidopsis s ems wi h educed hypoco yl diame e and
ascula cell numbe in he cycd3;1-3 iple mu an gene a ed in
he Columbia backg ound (Collins e al., 2015), al hough he
con ibu ion o indi idual CYCD3 genes was no de e mined. This
suppo s a scena io in which CYCD3s a e co e egula o s o cambial
cell p oli e a ion in bo h shoo s and oo s. Indeed, ou exp ession
da a sugges ha CYCD3s could play a ole in oo seconda y
g ow h. We compa ed he s ele c oss-sec ional a ea in cycd3;1 o
ha o WT immedia ely unde he hypoco yl du ing seconda y
g ow h (Fig. 1A). In o de o a oid con ounding e ec s om o he
polymo phisms in he Le and Col-O backg ounds, we analysed he
cycd3;1 allele in he Le backg ound, in which i was ini ially
gene a ed (Pa ino e al., 1999). A 17 days a e ge mina ion
(DAG), cycd3;1 oo s displayed a na owe s ele han WT
coun e pa s (Fig. 1C). Concomi an wi h educed cell di ision
ac i i y, cycd3;1 oo s had a educed numbe o ascula cells
(supplemen a y ma e ial Fig. S1). We conclude ha CYCD3;1
p omo es oo seconda y g ow h. Roo diame e was educed o a
simila ex en in bo h cycd3;1 and cycd3;1-3 iple mu an s in he
Le backg ound (supplemen a y ma e ial Fig. S2A,B), consis en
wi h CYCD3;1 being p ima ily equi ed among he CYCD3s genes
o seconda y g ow h in Le oo s.
ANT con ibu es o oo seconda y hickening
To iden i y po en ial egula o s o CYCD3;1 exp ession, we
pe o med genome-wide Pea son’s co ela ion es s o CYCD3;1
coexp ession wi h all known ansc ip ion ac o s ac oss >500
public A yme ix ATH1 mic oa ay da ase s anno a ed as
conduc ed on oo issues. Exp ession o CYCD3;1 was mos
highly co ela ed wi h AINTEGUMENTA (ANT) (supplemen a y
ma e ial Table S2). ANT is a membe o he AP2 (APETALA2)/
EREBP (E hylene Response Elemen Binding P o ein) amily, and
alls in o he ANT-lineage o he AP2-like subg oup (Kim e al.,
2006). Se e al membe s o he AP2-like subg oup a e associa ed
wi h de elopmen al egula ion du ing g ow h o young issues
(Nole-Wilson e al., 2005). ANT is in ol ed in he con ol o la e al
ae ial o gan size ia he egula ion o cell p oli e a ion (Mizukami
and Fische , 2000), and is associa ed wi h ui g ow h in apple
(Dash and Malladi, 2012) and seasonal bud do mancy in hyb id
aspen (Ka lbe g e al., 2011). Mo eo e , ec opic exp ession o ANT
in lea es inc eased le els o CYCD3;1 mRNA (Mizukami and
Fische , 2000). The e o e, ANT is a candida e egula o o
CYCD3;1 du ing seconda y oo g ow h.
Fig. 1. CYCD3;1 is exp essed in he oo
cambium and egula es seconda y
g ow h. (A) T ans e se sec ions o oo s
aken immedia ely below he hypoco yl a
ime poin s shown. A owheads indica e
ecen ly o med cell walls. (B) Exp ession
pa e ns o CYCD3 p omo e :GUS epo e s.
pCYCD3;1 d i es GUS exp ession in he
cambium and he pe icycle (le panel),
whe eas pCYCD3;2 and pCYCD3;3 d i e
GUS exp ession in he cambium and phloem
(middle and igh panel; ed a ows).
(C) S ele c oss-sec ional a ea in 17 DAG Le
and cycd3;1 oo s. ****P<0.0001; E o ba s
ep esen s.e.m. (D) T ans e se sec ions o
oo s in C. Scale ba s: 100 µm.
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To es his hypo hesis, he gene ic in e ac ion be ween ANT and
CYCD3;1 was in es iga ed. The o iginal an -9 allele isola ed in he
Landsbe g e ec a eco ype was in es iga ed. No s a is ically
signi ican educ ion in oo c oss-sec ional a ea was obse ed in
an -9 mu an s (Fig. 2A,B). Howe e , c oss-sec ional a ea was
educed o a g ea e ex en in an -9 cycd3;1 double mu an s han in
he cycd3;1 single mu an (Fig. 2A,B), sugges ing a con ibu ion o
ANT o seconda y hickening h ough a syne gis ic gene ic
in e ac ion be ween ANT and CYCD3;1. To u he in es iga e he
con ibu ion o ANT o seconda y g ow h, we iden i ied a new an
mu an in he Col-0 backg ound de i ed om he GABI-Ka
collec ion (supplemen a y ma e ial Fig. S3A). Homozygous
an -GK plan s display he cha ac e is ic an mu an pheno ypes o
educed lo al o gan size (supplemen a y ma e ial Fig. S3A) and ail
o p oduce seeds. In e es ingly, oo c oss-sec ional a ea and
ascula cell numbe we e educed in an -GK mu an s o a simila
ex en as he educ ions seen in cycd3;1 mu an s (Fig. 2C,D and
supplemen a y ma e ial Fig. S1).
To con i m ha e ec s o loss o unc ional ANT and CYCD3;1
in he shoo did no in luence o cause he oo pheno ypes desc ibed
he e, we g a ed WT shoo scion on o an -GK and cycd3;1 oo
scion. The pheno ypes emained (supplemen a y ma e ial Fig. S4),
con i ming ha hese a e independen oo pheno ypes.
Fu he mo e, oo elonga ion was no a ec ed by an and cycd3;1
mu a ions (supplemen a y ma e ial Fig. S6), demons a ing ha he
seconda y g ow h pheno ype was no caused by al e ed oo
on ogeny dynamics. The an -GK allele is in he Col-0 backg ound,
whe eas he an -9 allele is in Le , possibly explaining he di e ence
Fig. 2. In e ac ion be ween ANT and CYCD3;1 in oo seconda y g ow h. (A,C) S ele c oss-sec ional a ea in 30 DAG Le , cycd3;1,an -9 and
an -9 cycd3;1 oo s (A) and 21 DAG Col-0, an -GK and cycd3;1 (Col-0) oo s (C). *0.01<P<0.05, **0.001<P<0.01, ****P<0.0001; E o ba s ep esen s.e.m.
(B,D) T ans e se sec ions o oo s in A (B) and C (D). Scale ba s: 100 µm. (E) T ans e se sec ion om a GUS-s ained 14 DAG pANT:GUS oo . Scale ba : 50 μm.
(F) pCYCD3;1:GUS-GFP exp ession in 14 DAG ANT (i) and an -GK (ii) oo s. Scale ba : 200 µm. (G) qPCR analysis o CYCD3;1 and GFP in 16 day-old ANT
pCYCD3;1:GUS-GFP and an -GK pCYCD3;1:GUS-GFP oo s. E o ba s: s.d. in ou biological eplica es, each om eigh pooled oo s.
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in he se e i ies o hese pheno ypes. We conclude ha ANT also
egula es oo seconda y hickening.
Rela i ely high le els o ANT mRNA ha e been epo ed in oo s
(Ellio e al., 1996), bu he oo exp ession pa e n emains
unknown. Exp ession o a pANT:GUS epo e was eadily de ec ed
in he oo cambium (Fig. 2E). Suppo ing his, in a ansgenic line
exp essing a his one H2B-YFP usion unde he con ol o he ANT
p omo e ( pANT:H2B-YFP), ela i ely s ong luo escence was
obse ed in he s ele o he mo e ma u e oo (supplemen a y
ma e ial Fig. S6).
Regula ion o CYCD3;1 by ANT in shoo o gans has been
p oposed bu no demons a ed (Anas asiou and Lenha d, 2007;
Ho iguchi e al., 2009; Sch u e al., 2006). To es whe he ANT
migh egula e CYCD3;1 exp ession in he oo , a pCYCD3;1:GUS-
GFP epo e was in oduced in o he an -GK mu an . Visual
compa ison o pCYCD3;1:GUS-GFP exp ession in sibling F3
an -GK and WT plan s did no e eal a educ ion in exp ession in
an -GK homozygous oo s (Fig. 2F). Howe e , qPCR analyses o
oo mRNA e ealed a small educ ion o bo h na i e CYCD3;1 and
GUS-GFP ansc ip abundance in an -GK pCYCD3;1:GUS-GFP
mu an s (Fig. 2G). This could howe e be explained by a ela i e
dec ease in he abundance o CYCD3;1-exp essing cambial cells in
he an -GK mu an . The e o e, whils i emains possible ha ANT
egula es CYCD3;1, aken oge he wi h he gene ic e idence no
s ong egula ion is indica ed. Suppo ing his conclusion,
CYCD3;1 ansc ip abundance was no educed in an -9 oo s
(supplemen a y ma e ial Fig. S7). Fu he mo e, an addi i e
pheno ype in he an -9 cycd3;1 double mu an was also shown o
A abidopsis pe al epide mal cell size, and CYCD3;1 exp ession was
no educed in young an -9 lowe s (Randall e al., 2015).
Cy okinin signalling egula es bo h ANT and CYCD3;1
exp ession
an , cycd3;1 and cy okinin syn hesis/ esponse mu an s show
common pheno ypes o de ec i e oo seconda y hickening
(Heja ko e al., 2009; Ma sumo o-Ki ano e al., 2008). Since
cy okinins a e known o egula e CYCD3;1 ac i i y in shoo issues
(Dewi e e al., 2007), we assessed cy okinins as po en ial egula o s
o ANT and CYCD3;1 in he oo cambium.
We i s analysed hei exp ession in esponse o exogenous
cy okinin. qRT-PCR analysis o oo mRNA showed ha ollowing
cy okinin applica ion o plan s 14 DAG, ANT ansc ip le els we e
inc eased 6- old ela i e o un ea ed plan s, co ela ing wi h a
smalle inc ease in CYCD3;1 ansc ip le els (supplemen a y
ma e ial Fig. S8A). Suppo ing induc ion o CYCD3;1 by
cy okinins, pCYCD3;1:GUS exp ession was also induced in oo s
a e addi ion o he syn he ic cy okinin kine in (supplemen a y
ma e ial Fig. S8B).
To analyse induc ion o ANT and CYCD3;1 wi h g ea e
esolu ion, we measu ed exp ession a se e al ime poin s
ollowing addi ion o cy okinins o ip 1,3,5,7 mu an s. These
mu an s ca y loss-o - unc ion alleles o ou ip genes (ip 1,3,5,7),
which encode isopen enyl ans e ases in ol ed in cy okinin
biosyn hesis. These mul iple mu an s ha e educed le els o
isopen enyladenine as well as ans-zea in ( Z), a cy okinin shown
o ha e an e ec on cambium p oli e a ion (Ma sumo o-Ki ano
e al., 2008; Miyawaki e al., 2006). Since hese mu an s ail o
unde go p ope seconda y g ow h in oo s (Ma sumo o-Ki ano
e al., 2008), he use o his mu an should limi he amoun o
backg ound ANT and CYCD3;1 exp ession. Consis en wi h
cambium-exp ession o ANT and CYCD3;1, exp ession o hese
genes was educed in he ip 1,3,5,7 mu an (Fig. 3A). Ele a ed ANT
and CYCD3;1 exp ession was obse ed om 4 h a e incuba ion
wi h BAP (S uden ’s - es , P<0.05; n=3 in each case), inc easing
un il 24 h.
We hen in es iga ed he cy okinin equi emen o he inc eased
ANT p omo e ac i i y obse ed du ing he ansi ion om p ima y
o seconda y oo g ow h (Fig. 3B). We analysed exp ession o
pANT:H2B-YFP in ip 1,3,5,7 mu an s. Du ing he ac i a ion s age
a ound 5 DAG (Fig. 1A), when he i s cell di ision e en in he
p ocambium occu s, weak YFP signal om he pANT:H2B-YFP
cons uc was de ec ed in he oo s o bo h WT and ip 1;3;5;7 plan s
(Fig. 3B op). Quan i a ion o he YFP signal om con ocal images
showed simila signal in ensi y in bo h WT and ip mu an plan s
(Fig. 3B le ). Howe e , whe eas in WT plan s he signal in ensi ied
in oo s du ing he ansi ion s age (7 DAG), when p ocambial cells
begin o p oli e a e, in ip 1;3;5;7 oo s i did no (Fig. 3B). We
sugges ha he inc ease o ANT exp ession obse ed in WT oo s
depends on no mal le els o cy okinins. Al e na i ely, ANT
exp ession migh be delayed due o a delay in ascula issue
de elopmen in he ip 1;3;5;7 mu an .
To u he in es iga e he dependence o ANT and CYCD3;1
exp ession on cy okinin, we used p35S:CKX1 plan s o e exp essing
cy okinin oxidase leading o lowe le els o cy okinins (We ne
e al., 2001). These displayed educed abundance o ANT and
CYCD3;1 ansc ip s in oo s (Fig. 3C). Taken oge he , hese
esul s s ongly indica e egula ion o ANT and CYCD3;1 in he oo
ascula issue by cy okinins.
ANT and CYCD3;1 a e in ol ed in he egula ion o oo
seconda y g ow h by cy okinins
To de e mine whe he ANT and CYCD3;1 a e pa o he signalling
mechanism by which cy okinins p omo e seconda y hickening in
oo s, he oo hickening esponse o cy okinins was analysed in
mu an s. Ini ially, he esponse o cycd3;1 oo s o cy okinins was
compa ed o WT. Al hough cycd3;1 oo s we e hinne han WT and
emained so wi h low concen a ions o Z, highe concen a ions o
Z es o ed seconda y hickening o a le el compa able wi h WT
(supplemen a y ma e ial Fig. S9). We nex compa ed his esponse
in WT, an -9, cycd3;1, and an -9 cycd3;1 double mu an s. As
p e iously obse ed (Fig. 2A and supplemen a y ma e ial Fig. S7),
wi hou addi ion o cy okinins cycd3;1 and an -9 cycd3;1 oo s
showed educed diame e compa ed o WT Le oo s, he double
mu an being hinne han he single mu an (Fig. 3D). All
geno ypes espond o Z by inc easing in diame e bu no ably he
an -9 cycd3;1 double mu an emains hinne han o he geno ypes
a highe Z concen a ions (1000 ng/µl; Fig. 3D), suppo ing a
syne gis ic in e ac ion be ween ANT and CYCD3;1 and implying
ha hese ac o s independen ly con ibu e o adial cell di ision
ac i i y in he cambium. This da a also indica es ha o he ac o s
can media e he esponse o he cambium o cy okinins in he
absence o ANT and CYCD3;1.
Conse ed oles o ANT and CYCD3;1 in seconda y g ow h
Bo h ANT and CYCD3 genes a e widely conse ed amongs
angiospe ms (Kim e al., 2006; Menges e al., 2007) and
exp ession o he popla ANT o hologue has been epo ed in
cambial issue (Sch ade e al., 2004; Zhang e al., 2011). I was also
epo ed ha down- egula ion o ANT and CYCD3 o hologues in
hyb id aspen (P. emula x emuloides) is equi ed o bud-g ow h
cessa ion in his species (Ka lbe g e al., 2011). We analysed he
ac i i y o he popla ANT o hologue AIL1 in s ems unde going
seconda y g ow h, and obse ed ac i i y wi hin he cambium
(Fig. 4A), consis en wi h a po en ial ole o AIL1 in he egula ion
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o seconda y g ow h in his species. The ac i i y o a p omo e
sequence designa ed P ANT was also ecen ly epo ed in he
cambium (E chells e al., 2015); his p omo e sequence was de i ed
independen ly bu appea s o be ha o P AIL1 acco ding o he
locus iden i y, suppo ing he exp ession pa e n desc ibed he e. As
cy okinins also egula e seconda y g ow h in popla (Nieminen
e al., 2008), we analysed he exp ession o P AIL1 and he popla
CYCD3 homologue P CYCD3;2 ollowing cy okinin ea men s. I
should be no ed ha he CYCD3 gene numbe su ixes deno e
a bi a y o de o naming in ha species (Renaudin e al., 1996).
qRT-PCR analyses e ealed inc eases in ela i e abundances o bo h
P AIL1 and P CYCD3;2 ansc ip s a e wel e hou s o cy okinin
ea men (Fig. 4B). The e o e, we p opose ha he egula ion o
seconda y g ow h by cy okinin-induced ANT and CYCD3;1 is
conse ed in highe plan s.
Conclusions
The ansc ip ion ac o ANT and he cyclin CYCD3;1 play
independen oles in egula ing cell di ision du ing seconda y
g ow h o A abidopsis oo s. cycd3;1,an and cy okinin
biosyn hesis and ecep o mu an s sha e a common ailu e in
cambial p oli e a ion, and we show ha hese componen s ac in
conse ed pa hways linking cy okinins o de elopmen ally
egula ed cell p oli e a ion.
Fig. 3. ANT and CYCD3;1 espond o cy okinins in
oo seconda y hickening. (A) qPCR analysis o
ARR5, ANT and CYCD3;1 in Col-0 and ip 1;3;5;7 oo s
ea ed wi h DMSO and ip 1;3;5;7 oo s ea ed wi h 1 µM
BAP o he pe iods indica ed. E o ba s ep esen s.d.
om 3 biological eplica es. (B) In ensi y o YFP signal in
lines exp essing pANT:H2B-YFP a 5 DAG and 7 DAG in
WT plan s s ip 1,3,5,7 plan s. WT1-3 and ip 1-3 each
ep esen h ee independen T3 lines homozygous o
pANT:H2B-YFP.ip 1-3 a e also homozygous o
ip 1;3;5;7 alleles. In each line, signal in ensi y was
measu ed om >50 nuclei. F equency dis ibu ions o
signal in ensi y a e shown. (C) T ansc ip le els o
CYCD3;1 and ANT in Col-0 and p35S:CKX1 oo s. s.d. in
h ee biological eplica es is shown. (D) Diame e o Le ,
cycd3;1, an -9 and an -9 cycd3;1 oo s ollowing Z
ea men s. Roo s we e g own o 11 days hen
ans e ed o media con aining (o no o con ol) Z.
E o ba s ep esen s.e.m.
1233
RESEARCH ARTICLE Biology Open (2015) 4, 1229-1236 doi:10.1242/bio.013128
Biology Open

MATERIALS AND METHODS
Plan ma e ial and g ow h
A abidopsis plan s we e g own in 16 h days a 22°C on MS medium
con aining 1.5% suc ose, 0.5 g/l 2-(N-mo pholino)e hanesul onic acid and
1% aga . CYCD3 p omo e :GUS epo e lines, cycd3;1, an -9, ip 1;3;5;7
and c e2;3;4 mu an s ha e been desc ibed (Dewi e e al., 2007; Ellio e al.,
1996; Higuchi e al., 2004; Miyawaki e al., 2006; Mizukami and Fische ,
2000). The cycd3;1 mu an ini ially isola ed om a Landsbe g e ec a (Le )
DS elemen inse ion lib a y (Pa ino e al., 1999) was backc ossed wice o
Le WT, and he iple cycd3;1-3 mu an in he Le backg ound was
gene a ed by c ossing cycd3;1 wi h cycd3;2, and backc ossing lines
hemizygous o bo h alleles o Le wice be o e in og essing he cycd3;3
allele om he EXOTIC collec ion (Dewi e e al., 2007). The an -GK
mu an (GK-874H08; TAIR accession no.: 1006453905) is desc ibed in
supplemen a y ma e ial Fig. S3. The pCYCD3;1:GUS-GFP and pANT:
His one2b-YFP (pANT:H2B-YFP) epo e s we e cons uc ed in
pKGWFS7 (Ka imi e al., 2002). 947 bp o DNA ups eam ( ep esen ing
sequence o he adjacen gene) o he CYCD3;1 s a codon was used.
5137 bp o ANT ups eam sequence was used o pANT:H2B-YFP, and
6291 bp o pANT:GUS which was isola ed om a lambda EMBL3 lib a y
(Da id Smy h and B ian Kwan, Monash Uni e si y, Melbou ne, Aus alia).
Scion o A ip 1;3;5;7 was g a ed on o WT s ock and used o lo al dipping
(Clough and Ben , 1998).
WT Populus emula x emuloides line T89 was used o qPCR analyses.
P. emula x emuloides seedlings we e g own o one mon h in long-day
g eenhouse condi ions a 22°C in 5 li e po s. The pAIL1:GUS line has been
desc ibed (Ka lbe g e al., 2011).
A abidopsis mic og a ing
A abidopsis plan s we e g a ed acco ding o a published p o ocol
(Tu nbull e al., 2002) wi h he ollowing modi ica ions. E hanol
s e ilised A abidopsis seeds we e ge mina ed on 1/2 Mu ashige and
Skoog (MS) medium plus 1% Di co aga (pH 5.7; 1% suc ose) and g own
on e ically-moun ed Pe i dishes unde long (16 h o 80–100 µmoles
ligh , Col-0 and an -GK g a s) o sho day condi ions (8 h o
80–100 µmoles ligh , Le and cycd3;1 g a s) a 20°C. G a ing was
pe o med unde s e ile condi ions in a lamina low hood wi h a Zeiss
po able dissec ing mic oscope. 5–6 day-old seedlings we e ans e ed o
9 cm Pe i dishes ha con ained one laye o 2.5×4 cm s e ilised Hybond N
memb ane (GE Heal hca e) on op o wo s e ilised 8 cm disks o 3 mm
Ch Wha man pape (Scien i ic Labo a o y Supplies). The Wha man
pape and Hybond N memb ane we e kep mois using a 1% suc ose
solu ion (Col-0 and an -GK g a s) o s e ile dis illed wa e (Le and
cycd3;1 g a s). A ans e se cu was made h ough he hypoco yl close o
he shoo using a ascula dissec ing kni e (Ul a Fine Mic o Kni e; Fine
Science Tools). In addi ion, one co yledon was emo ed o assis in
aligning he g a ed pieces. In he case o sel -g a s, an addi ional 1 mm
segmen was cu om he hypoco yl and disca ded. G a s we e assembled
by bu alignmen o he wo cu hal es wi h no suppo ing colla . A e
g a ing, Pe i dishes we e sealed wi h pa a ilm, moun ed e ically unde
long (Col-0 and an -GK g a s) o sho day condi ions (Le and cycd3;1
g a s) and moni o ed o 7 days a 20°C. A e wa ds, success ul g a s and
un-g a ed con ols we e ans e ed o 1/2 MS pla es wi h 1% suc ose
unde long day condi ions a 20°C o addi ional 2–3 weeks. Fo an -GK
ela ed g a s and un-g a ed plan s, DNA was ex ac ed and geno yping
PCR was pe o med o con i m he geno ype o each scion and s ock.
Roo s om s ock we e sampled and embedded wi h Leica esin. 5 µm- hin
plas ic sec ions we e cu a 5 mm below he hypoco yl- oo junc ion and
s ained wi h Toluidine Blue O.
Mic oscopy and ana omical analyses
T ans e se sec ioning and GUS assays we e desc ibed (Mahonen e al.,
2006, 2000; Nieuwland e al., 2009). Sec ions we e aken wi hin 1 cm o he
oo -hypoco yl junc ion. Mic oscopy was pe o med using a Zeiss LSM 710
o a Leica SP5. Roo s we e s ained wi h 100 μg/ml p opidium iodide.
Cy okinin induc ion and quan i a i e PCR
BAP, kine in and ans-zea in we e dissol ed in dime hyl sul oxide and
dilu ed in s e ile wa e . 2iP was dissol ed in 20 mM NaPi bu e .
A abidopsis induc ions we e pe o med by ansplan ing on o pla es
con aining cy okinins o equi alen con ol bu e . Induc ions in
P. emula x emuloides seedlings we e pe o med by subme ging s em
pieces in solu ions indica ed. Col-0 and ip 1,3,5,7 mu an s we e g own on a
nylon mesh (SEFAR NITEX 03-100/44) on e ical pla es con aining
0.8% Plan Aga (Duche a), 1% suc ose (Duche a), 0.5× MS medium
including i amins (Duche a) and pH adjus ed o 5.7–5.8wi h20×MES
bu e (Mes monohyd a e, Duche a). 7-day-old plan s we e ans e ed
wi h he mesh o pla es con aining 1 µM BAP o he espec i e DMSO
con ol. P ima y oo samples consis ing o 40 plan s we e ha es ed a 1, 2,
4, 6, 8 and 24 h a e he ans e by cu ing a ew millime e s below he
hypoco yl, p ima y oo ips and la e al oo s we e disca ded. RNA was
isola ed wi h Qiagen RNeasy Plan Mini Ki wi h an on-column DNase
ea men . Addi ional DNase ea men wi h DNase I (RNase- ee)
(The mo Scien i ic) was pe o med o 1 µg o RNA p io o he oligo-
dT-p imed cDNA syn hesis wi h Fi s S and cDNA Syn hesis Ki
(The mo Scien i ic). Exp ession o ANT,CYCD3;1 and ARR5 was
quan i ied wi h p ime s lis ed in supplemen a y ma e ial Table S3wi h
HOT FIREPol
®
E aG een
®
qPCR Mix Plus (no ROX) (Solis Biodyne).
The Bio-Rad CFX384 was used wi h one cycle 95°C o 15 min, 40 cycles
each consis ing o 95°C o 15 s, 60°C o 30 s and 72°C o 30 s, one cycle
95°C o 10 s ollowed by mel cu e analysis. Raw da a alues we e
no malized o he geome ic mean o ou con ol genes (Vandesompele
e al., 2002) and old changes calcula ed in compa ison o he Col-0
exp ession le el. The expe imen was pe o med in iplica e.
Fo o he expe imen s, RNA was isola ed using T iPu e (Roche
Diagnos ics) and cDNA was syn hesized using he Ambion Re osc ip
ki . qRT-PCR analyses we e pe o med as desc ibed o A abidopsis
(Menges and Mu ay, 2002) and P. emula x emuloides (Nieminen e al.,
Fig. 4. Popla ANT is exp essed in he cambium and is induced by cy okinins. C oss-sec ion o a Popla pAIL1:GUS s em ollowing GUS assay (A) and
qPCR analyses o P ANT and P CYCD3;2 ansc ip s ollowing ea men s o cy okinin (100 nM 2iP) o mock ea men s o one o wel e hou s (B). Two
indi iduals ep esen ed sepa a ely in adjacen ba s we e used o he expe imen . T ansc ip le els we e no malized o P TUA2. E o ba s: s.d. in ou echnical
eplica es.
1234
RESEARCH ARTICLE Biology Open (2015) 4, 1229-1236 doi:10.1242/bio.013128
Biology Open
2008).Gene-speci ic p ime s a e lis ed (supplemen a y ma e ial Table S1).
Rela i e ansc ip le els we e quan i ied using he ΔΔCT me hod (Li ak
and Schmi gen, 2001).
S a is ics
S uden ’s - es s we e pe o med in G aphPad P ism. *0.01<P<0.05
**0.001<P<0.01 ***0.0001<P<0.001 ****P<0.0001. Pea son’s co ela ion
es s we e ca ied ou in R ( -p ojec .o g).
Acknowledgemen s
We a e mos g a e ul o Da id Smy h and B ian Kwan who made he pANT:GUS line,
Te a Ve noux o discussions, and Angela Ma chbank, Jo Kilby, Ka ja Kainulainen
and Mikko He pola o excellen echnical suppo .
Compe ing in e es s
The au ho s decla e no compe ing o inancial in e es s.
Au ho con ibu ions
RSR w o e he manusc ip . RSR, SM & WD analysed da a. RSR, SM & AA
pe o med qPCR expe imen s. RSR, SM, JZ, AE & AK c ea ed and analysed issue
c oss-sec ions. TB conduc ed he ime-cou se cy okinin induc ion expe imen in
A abidopsis. RSR & KB conduc ed geno yping o an -9 cycd3;1 mu an s. CWM
conduc ed mic og a ing. AA, CF, MMK & JYL c ea ed ansgenic lines and
conduc ed p elimina y expe imen s. JI conduc ed qPCR on Popla -de i ed cDNA.
TK, APH, RB, WD, YH & JAHM con ibu ed in ellec ually o p ojec planning and
manusc ip p epa a ion.
Funding
Wo k in he J.A.H.M. lab was suppo ed by g an s BB/D011914 om he
Bio echnology and Biological Sciences Resea ch Council and a g an om he
Eu opean Resea ch A ea in Plan Genomics o J.A.H.M. (BB/E024858) and Y.H.,
who was also suppo ed by he Academy o Finland and Tekes.
Supplemen a y ma e ial
Supplemen a y ma e ial a ailable online a
h p://bio.biologis s.o g/lookup/suppl/doi:10.1242/bio.013128/-/DC1
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