Cu en Biology 23, 804–809, May 6, 2013 ª2013 Else ie L d All igh s ese ed h p://dx.doi.o g/10.1016/j.cub.2013.03.053
Repo
Dynamic Regula ion o Co ical
Mic o ubule O ganiza ion
h ough P e oldin-DELLA In e ac ion
An onella Locascio,
1,2
Miguel A. Bla
´zquez,
1,
*
and Da id Alabadı
´
1
1
Ins i u o de Biologı
´a Molecula y Celula de Plan as,
Consejo Supe io de In es igaciones Cien ı
´ icas–Uni e sidad
Poli e
´cnica de Valencia, Ingenie o Faus o Elio s/n,
46022 Valencia, Spain
2
Depa men o En i onmen al Ag onomy and C op
P oduc ion (DAFNAE), Uni e si y o Pado a, Viale
dell’Uni e si a
`16, 35020 Legna o-Pado a, I aly
Summa y
Plan mo phogenesis elies on speci ic pa e ns o cell
di ision and expansion. I is well es ablished ha co ical
mic o ubules in luence he di ec ion o cell expansion
[1, 2], bu less is known abou he molecula mechanisms
ha egula e mic o ubule a angemen . He e we show ha
he phy oho mones gibbe ellins (GAs) egula e mic o ubule
o ien a ion h ough physical in e ac ion be ween he
nuclea -localized DELLA p o eins and he p e oldin com-
plex, a cochape one equi ed o ubulin olding [3]. In he
p esence o GA, DELLA p o eins a e deg aded, and he p e-
oldin complex s ays in he cy oplasm and is unc ional. In
he absence o GA, he p e oldin complex is localized o
he nucleus, which se e ely comp omises a/b- ubulin he e -
odime a ailabili y, a ec ing mic o ubule o ganiza ion. The
physiological ele ance o his molecula mechanism was
con i med by he obse a ion ha he daily hy hm o
plan g ow h was accompanied by coo dina ed oscilla ion
o DELLA accumula ion, p e oldin subcellula localiza ion,
and co ical mic o ubule eo ien a ion.
Resul s and Discussion
Hypoco yl elonga ion is one o he simples models in which o
s udy plan mo phogenesis. G ow h o his o gan is almos
exclusi ely sub ended by aniso opic cell expansion [4], and
cell g ow h di ec ion is de e mined in pa by he o ien a ion
o co ical mic o ubules a he inne angen ial wall o epi-
de mal cells, h ough hei in luence on he deposi ion o cell
wall ma e ial [2, 5, 6]. Despi e he obse a ions ha sugges
ha i is possible o uncouple aniso opic g ow h om he
ea angemen o co ical mic o ubules [7, 8], mo e ecen
e idence has con i med he key ole o mic o ubule o ganiza-
ion when his is p ecisely analyzed in expanding cells wi hin
he hypoco yl [1]. Among he signals ha egula e mic o ubule
dynamics, he phy oho mones gibbe ellins (GAs) p omo e cell
expansion and also di ec he o ien a ion o he co ical mic o-
ubule a ay pe pendicula o he g ow h axis [1, 7, 9], aising
he ques ion o how hese wo p ocesses a e coo dina ed [10].
DELLAs a e nuclea p o eins ha media e ansc ip ional
egula ion o cell expansion genes by GAs [11] and o he
p ocesses along plan de elopmen [12–17]. In A abidopsis,
DELLAs a e encoded by i e genes (GAI,RGA,RGL1,RGL2,
and RGL3). They unc ion as ansc ip ional egula o s and,
al hough hey lack any known DNA-binding domain, his
ole is exe ed h ough he in e ac ion wi h o he p o eins, in
pa icula , ansc ip ion ac o s [18]. This abili y o es ablish
physical in e ac ions also p o ides he cells wi h a mechanism
o c oss alk be ween signaling pa hways. Fo ins ance, he
opposi e e ec o ligh and GAs on cell expansion is modu-
la ed by he in e ac ion be ween DELLA p o eins—whose
s abili y is dec eased by he ho mone—and PIF (phy o-
ch ome-in e ac ing p o ein) ansc ip ion ac o s—which a e
des abilized by ligh [11, 19]. Howe e , his single molecula
mechanism does no explain how aniso opic cell expansion
is egula ed by GAs.
To iden i y he pa ne s h ough which DELLA p o eins exe
hei egula o y unc ions, we pe o med a yeas wo-hyb id
sc eening using a unca ed e sion o GAI ha p e en s ac i-
a ion o he epo e genes, as shown o RGA, a ela ed
DELLA p o ein [11, 20]. Two o he posi i e clones encoded
ull-leng h e sions o p e oldin5 (PFD5), one o he subuni s
o a he e ohexame ic molecula cochape one, conse ed
om A chaea o euka yo es [21, 22]. In e ac ion ex ended
also o PFD3, bu no o he o he membe s o he PFD complex
(Figu e 1A; see also Figu e S1 a ailable online), indica ing ha
he in e ac ion wi h GAI was es ic ed o he a-subuni s, which
a e loca ed a he co e o he complex [21]. Coimmunop ecipi-
a ion s udies in in il a ed Nico iana ben hamiana lea es
u he co obo a ed his in e ac ion in plan a (Figu e 1B). Gi en
ha DELLA p o eins a e localized in he nucleus [23], whe eas
he chape one unc ion o PFD is equi ed in he cy osol [3],
we examined he subcellula localiza ion o he GAI-PFD5 in e -
ac ion by bimolecula luo escence complemen a ion (BiFC) in
N. ben hamiana lea es [24, 25]. In e es ingly, signal om he
econs i u ed yellow luo escen p o ein (YFP) was isible in
nuclei o coin il a ed cells (Figu e 1C), sugges ing ha he
in e ac ion wi h DELLAs migh o ce he accumula ion o
PFD5 in he nucleus. This idea was u he suppo ed by he
obse a ion ha YFP-PFD5 was de ec ed in he cy osol o
N. ben hamiana lea es (Figu e 1D), whe eas i mos ly appea ed
in he nucleus when coexp essed wi h RFP-GAI (Figu e 1E).
To con i m ha he nuclea accumula ion o PFD5 occu s in
wild- ype A abidopsis plan s in a DELLA-dependen manne ,
we in es iga ed he localiza ion o PFD5 in da k-g own p d5
null mu an seedlings complemen ed wi h a cons uc ex-
p essing PFD5-GFP unde he con ol o he PFD5 p omo e
(see Supplemen al Expe imen al P ocedu es). As expec ed,
in un ea ed, con ol seedlings, PFD5-GFP appea ed in he
cy osol o cells loca ed in he op hi d o e iola ed hypoco yls
(Figu e 2A), which co espond o ac i ely expanding, GA-
esponsi e cells [4]. Howe e , incuba ion wi h paclobu azol
(PAC), an inhibi o o GA biosyn hesis ha induces he accu-
mula ion o DELLA p o eins [23], caused he accumula ion o
PFD5-GFP in he nucleus, whe e GFP-RGA accumula es in
he con ol epo e line [26]. Impo an ly, cy osolic localiza ion
was es o ed 3 h a e applica ion o GA
4
o PAC-g own
seedlings, concomi an ly wi h he disappea ance o GFP-
RGA om nuclei, indica ing ha PFD5 localiza ion in he
nucleus equi es he con inuous p esence o DELLA p o eins.
Mo eo e , subcellula ac iona ion o whole seedling ex ac s
*Co espondence: mblazq[email p o ec ed]
showed ha PFD5 was p esen in he insoluble nuclea
ac ions o PAC- ea ed, wild- ype seedlings, whe eas i
appea ed in he cy osolic ac ion o seedlings o he pen uple
della mu an independen ly o he ea men (Figu e 2B). These
esul s indica e ha DELLA accumula ion is equi ed o he
localiza ion o PFD5 in he nucleus.
To assess whe he he in e ac ion be ween GAI and he
a-subuni s o PFD p o okes he accumula ion o he whole
PFD complex in he nucleus, we examined he subcellula
localiza ion o PFD6, a subuni ha does no in e ac di ec ly
wi h GAI (Figu es 1A, 1B, and S1). Using a ansgenic A abi-
dopsis line exp essing PFD6::PFD6-YFP (PFD6-YFP)[27], we
ound ha PFD6 showed he same beha io as PFD5-GFP
(compa e Figu es S2A and 2A). This esul sugges s ha he
in eg i y o he PFD complex is no al e ed by he GAI-PFD5
in e ac ion. This is also suppo ed by immunode ec ion o
PFD5 a e gel il a ion o na i e ex ac s o wild- ype seed-
lings g own in he p esence o PAC. Unde hese condi ions,
PFD5 appea ed in he ac ions co esponding o he in ac
PFD complex, simila ly o wha was obse ed in he ex ac s
o mock- ea ed seedlings (Figu e S2B). Rema kably, he
PFD complex elu ed in he same ac ion in PAC as in mock,
sugges ing ha DELLA p o eins a e no pe manen ly associ-
a ed o PFD o ha his in e ac ion is los du ing ex ac manip-
ula ion. The idea ha PFD localiza ion in he nucleus is
dynamic and in ima ely linked o DELLAs was con i med using
F1 seedlings o a c oss be ween a GAI::gai-1-GR line [28],
which exp esses a dominan e sion o he DELLA p o ein
ha is e ained in he cy osol unless he syn he ic glucoco i-
coid dexame hasone (DEX) is supplied, and he PFD6-YFP
line. As shown in Figu e 2C, PFD6-YFP was isible in he
cy osol o e iola ed hypoco yl epide mal cells, appea ed in
he nucleus 2 h a e DEX ea men , and was ully nuclea
6 h a e he applica ion o he glucoco icoid. Impo an ly,
he iming o he accumula ion o PFD6-YFP in he nucleus
coincides wi h he iming needed by gai-1-GR o exe i s an-
sc ip ional egula ion ac i i y [29, 30], which is consis en wi h
he idea ha nuclea accumula ion o he en i e PFD complex
depends upon he p esence o DELLAs.
The iden i ica ion o PFD3 and PFD5 as in e ac o s o DELLA
p o eins and he elocaliza ion o he cochape one complex
caused by his in e ac ion sugges ed a mechanism by which
GAs could egula e mic o ubule o ien a ion: he in e e ence
wi h PFD unc ion. In ac , A abidopsis null mu an s in a leas
PFD3,PFD5, and PFD6 a e de ec i e in PFD unc ion and
display a diso ganized a ay o co ical mic o ubules [22, 27]
equi alen o he one caused by GA de iciency (Figu e 3A).
PFD is equi ed o main ain app op ia e le els o ubulin
he e odime s in yeas [31], in animals [32], and in plan s [22].
The link be ween PFD ac i i y and mic o ubule a angemen
is based on wo pieces o e idence: (1) mic o ubule g ow h
a e and leng h a e di ec ly p opo ional o ubulin concen a-
ion among o he ac o s [33–35], and (2) co ical mic o ubule
ans e se a angemen in hypoco yl cells equi es long
mic o ubules [36]. Thus, we hypo hesized ha he DELLA-
dependen nuclea localiza ion o PFD could cause he disa -
angemen o co ical mic o ubules by educing he p ope
olding o ubulin p o eins in he cy osol, simila o he de ec s
ound in yeas gim mu an s a ec ed in PFD ac i i y [37–39]. To
es his hypo hesis, we analyzed a- and b- ubulin accumula-
ion in hei na i e con o ma ions by gel il a ion o ex ac s
o con ol and PAC- ea ed seedlings. As expec ed, mos o
a- ubulin was eco e ed in he he e odime ac ion o con ol
ex ac s, oge he wi h b- ubulin (Figu e 3B). Howe e , GA
de iciency caused by PAC- ea men p o oked mos o
a- ubulin o dissocia e om b- ubulin and appea in i s mono-
me ic o m (Figu e 3B). Simila ly, a la ge p opo ion o b- ubulin
shi ed om ubulin he e odime s o a lowe -size ac ion ha
could ep esen complexes wi h uniden i ied p o eins. In
summa y, ou esul s sugges ha DELLAs indi ec ly a ec
he polyme iza ion o mic o ubules (and ul ima ely hei abili y
o eo ien ) by con olling he a ailabili y o a/b- ubulin he e o-
dime s h ough, a leas , he p e en ion o PFD unc ion as a
ubulin cochape one in he cy osol.
Figu e 1. Physical In e ac ion be ween GAI and
Two Subuni s o he P e oldin Complex in he
Nucleus
(A) In e ac ion be ween GAI and he di e en sub-
uni s o he A abidopsis PFD complex by a yeas
wo-hyb id assay. L, Leu; T, T p; H, His; 3AT,
35 mM 3-amino iazole. ‘‘Vec o ’’ co esponds
o pDEST22.
(B) Coimmunop ecipi a ion o myc-GAI and YFP-
PFD5 exp essed in N. ben hamiana lea es. The
‘‘M5’’ e sion o myc-GAI (see Expe imen al
P ocedu es) was immunop ecipi a ed using
an i-myc conjuga ed pa amagne ic beads, and
GAI and PFD5 and PFD6 we e de ec ed in immu-
noblo s using an i-myc and an i-GFP. No e ha
PFD6 was no coimmunop ecipi a ed wi h GAI.
The sizes o he bands co espond o he ex-
pec ed sizes o he usion p o eins.
(C) BiFC analysis o he in e ac ion be ween GAI
and PFD5 in nuclei o N. ben hamiana lea cells.
Scale ba ep esen s 40 mm.
(D) Localiza ion o YFP-PFD5 in he cy osol o
cells o N. ben hamiana lea es when in il a ed
alone. Scale ba ep esen s 20 mm.
(E) Localiza ion o YFP-PFD5 in he nucleus
o a ep esen a i e N. ben hamiana lea cell
when coin il a ed wi h RFP-GAI. Scale ba ep e-
sen s 20 mm.
See also Figu e S1.
DELLA Regula ion o Mic o ubule O ganiza ion
805
The g ow h a e o A abidopsis hypoco yls oscilla es in a
daily ashion, wi h maximal g ow h occu ing a he end o
he nigh [40, 41]. The egula ion o DELLA p o ein s abili y
by he ci cadian clock is key o his oscilla o y beha io [42].
The e o e, i DELLAs coo dina ely egula e bo h expansion
and g ow h di ec ion, an impo an p edic ion is ha he sub-
cellula localiza ion o PFD and he p edominan o ien a ion
o co ical mic o ubules should be dic a ed by he phase o
DELLA accumula ion unde pho ope iodic condi ions. Indeed,
we ound ha mic o ubules in hypoco yl epide mal cells dis-
played a p e e ed o ien a ion pe pendicula o he cell expan-
sion axis du ing he g ow h phase owa d he end o he nigh
(ZT19) and andom o longi udinal sho ly a e dusk (ZT9) (Fig-
u es 4A and 4B), coinciding wi h he slowes g ow h a e. This
indica es ha he o ien a ion o he co ical a ay o mic o u-
bules is dynamic and subjec ed o diu nal egula ion. Mo e
impo an ly, diso ganized mic o ubule a angemen co e-
la ed wi h he accumula ion o GFP-RGA a ZT9 and he
nuclea accumula ion o PFD6-YFP, whe eas he ans e se
o ganiza ion coincided wi h he p esence o PFD6-YFP in he
cy osol (ZT19) and unde ec able GFP-RGA le els (Figu e 4A).
As expec ed, nuclea PFD6-YFP accumula ion a ZT9 was
no isible in seedlings con inuously g own in he p esence
o GAs (Figu e S3).
To gauge o wha ex en he DELLA-dependen daily
changes in PFD localiza ion a ec s he unc ion o he
complex, we examined o he PFD- egula ed p ocesses, o
ins ance he exp ession o ubulin genes, epo ed o be unde
Figu e 2. DELLA-Dependen Nuclea Accumula ion o PFD in A abidopsis
Hypoco yls
(A) Con ocal images o PFD5-GFP and GFP-RGA in he apical hi d o wild-
ype A abidopsis hypoco yls g own o 3 days in da kness in MS medium
(mock) o in MS supplemen ed wi h 1 mM PAC (‘‘pac’’ in igu e) and 3 h a e
he applica ion o 10 mMGA
4
o PAC-g own seedlings (pac + GA). Scale ba
ep esen s 40 mm.
(B) Immunochemical de ec ion o PFD5 and his one H3 p o eins in ac ion-
a ed ex ac s o A abidopsis seedlings. His one H3 was used as a ma ke o
he insoluble pa o he nuclea ac ion. C, cy osol; N-s, soluble ac ion o
he nucleus; N-i, insoluble ac ion o he nucleus. The sizes o he bands
co espond o he expec ed sizes o he co esponding p o eins (15 kDa
o PFD5, and 15–17 kDa o H3). The as e isk ma ks he band wi h he
p edic ed size when mul iple bands a e isible.
(C) Fluo escence o PFD6-YFP in a F1 seedling om a c oss wi h a GAI::gai-
1-GR line, be o e and 2 h and 4 h a e he applica ion o 10 mM dexa-
me hasone. Scale ba ep esen s 10 mm.
See also Figu e S2.
Figu e 3. DELLA-Dependen Regula ion o Co ical Mic o ubule A ange-
men and a/b-Tubulin He e odime s A ailabili y
(A) Con ocal images o co ical mic o ubules labeled wi h GFP in he
epide mal cells o he op hi d o hypoco yls o a TUA6-GFP line. Scale
ba ep esen s 10 mm. Quan i ica ion o he o ien a ion o co ical mic o u-
bules was ca ied ou in a leas 120 indi idual cells.
(B) Immunode ec ion o a- and b- ubulin in size- ac iona ed p o ein ex-
ac s a e gel ch oma og aphy. Figu es indica e he ac ion numbe .
In all panels, seedlings we e g own o 3 days in da kness in MS (mock) o in
MS supplemen ed wi h 1 mM PAC (‘‘pac’’ in igu e).
Cu en Biology Vol 23 No 9
806
he con ol o PFD in yeas [38]. Gi en ha he exp ession o
se e al TUA genes (encoding a- ubulin) in A abidopsis oscil-
la es in a daily ashion, acco ding o he DIURNAL da abase
[44], we examined he e ec o p d5 and pen uple della mu a-
ions in he cyclic beha io o hese genes. Consis en wi h he
obse a ion in yeas , exp ession o a- ubulin genes equi ed
he ac i i y o PFD5 (Figu e 4C) and, mo e in e es ingly, hei
daily oscilla ion was se e ely a ec ed in a pen uple della
mu an as well, suppo ing he idea ha DELLA-dependen
accumula ion o PFD in he nucleus is equi ed o he ex-
p ession o se e al TUA genes. In e es ingly, maximal daily
exp ession o he examined TUA genes did no coincide wi h
he pe iod o maximal g ow h a e, in ag eemen wi h he
idea ha ansla ional egula ion and olding o ubulin is a
c i ical s ep.
Ou esul s unde sco e he ole o DELLA p o eins as coo di-
na o s o dis inc p ocesses equi ed o plan g ow h and p o-
ide a molecula link be ween he iming, he execu ion, and
he di ec ion o cell expansion (Figu e 4D). The al e a ion in
PFD unc ion h ough p o ein-p o ein in e ac ion has also
been desc ibed in animal cells du ing i al in ec ion [45] and
may ep esen a s a egy o he con ol o ubulin a ailabili y.
Howe e , al hough a p ope ubulin concen a ion is a p e-
equisi e o he o ien a ion o co ical mic o ubules, addi ional
mechanisms ha e o be in oked o de e mine he inal o gani-
za ion o he mic o ubule a ays du ing cell expansion [46–48].
The DELLA-dependen localiza ion o PFD in he nucleus is a
he co e o his mechanism. I impai s he ac i i y o PFD as a
chape one in he cy osol, bu ou esul s also aise he
in iguing possibili y ha PFD may ha e an addi ional ole in
he nucleus. Nuclea localiza ion o PFD5 has been desc ibed
in animals, whe e i ac s as a egula o o he c-Myc ansc ip-
ion ac o [49–51]. I is he e o e possible ha he PFD com-
plex o i s subuni s pe o m a simila , DELLA-dependen ole
in egula ing ansc ip ion in plan cells.
Supplemen al In o ma ion
Supplemen al In o ma ion includes h ee igu es, Supplemen al Expe i-
men al P ocedu es, one able and can be ound wi h his a icle online a
h p://dx.doi.o g/10.1016/j.cub.2013.03.053.
Acknowledgmen s
We hank C. Some ille and Y. Gu o he PFD6-YFP line, J. Salinas o he
p d5 mu an , N. Ma ı
´n de la Rosa o he myc-GAI cons uc , and A. Fe ando
o he BiFC ec o s. We also hank S. Ta
´ aga o assis ance wi h he gel
il a ion assays and C. Fankhause , J. Salinas, and P. Rod ı
´guez o discus-
sions and c i ical eading o he manusc ip . This wo k was suppo ed by
g an s om he Spanish Minis y o Science and Inno a ion (BIO2010-
15071 and CSD2007-00057) and he Gene ali a Valenciana (ACOMP/
2011/288 and PROMETEO/2010/020). A.L. was unded du ing pa o he
wo k by Fondo pe gli In es imen i della Rice ca di Base o he I alian
Minis y o Educa ion, he Uni e si y, and Resea ch (MIUR).
Figu e 4. Tempo al Coo dina ion o PFD Accumula ion in he Nucleus and Co ical Mic o ubule A angemen
(A) Con ocal images o TUA6-GFP, PFD6-YFP, and GFP-RGA in he epide mal cells o he op hi d o hypoco yls o seedlings g own unde sho days (8 h o
ligh and 16 h o da kness). ZT, ime (in hou s) a e ligh s on. Scale ba ep esen s 20 mm.
(B) Quan i ica ion o he o ien a ion o co ical mic o ubules, which was ca ied ou in a leas 115 indi idual cells.
(C) Oscilla ion o TUA2,4,6 exp ession in seedlings g own unde sho -day cycles in wild- ype (Col-0) and p d5 mu an seedlings (le ) and in wild- ype (Le )
and pen uple della mu an seedlings ( igh ). Whi e and g ay ba s ep esen day and nigh , espec i ely. Da a poin s ep esen he mean 6SEM o h ee bio-
logical eplica es. The p ime s used in he RT-qPCR expe imen do no dis inguish be ween he h ee TUA genes, due o he high iden i y pe cen age o hei
nucleo ide sequence [43].
(D) Model o he coo dina ion by DELLAs o he h ee aspec s ha con o m o gan g ow h by cell expansion: empo al con ol o g ow h h ough he
oscilla ion o DELLA s abili y, con ol o cell expansion h ough physical in e ac ion be ween DELLAs and PIF (and possibly o he ) ansc ip ion ac o s,
and de e mina ion o g ow h di ec ion h ough physical in e ac ion be ween DELLAs and PFD wi h p edic able impac on he polyme iza ion o co ical
mic o ubules.
See also Figu e S3.
DELLA Regula ion o Mic o ubule O ganiza ion
807
Recei ed: Decembe 17, 2012
Re ised: Ma ch 6, 2013
Accep ed: Ma ch 22, 2013
Published: Ap il 11, 2013
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