Cytokinins act directly on lateral root founder cells to inhibit root initiation.

Abstract

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.

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Cytokinins Act Directly on Lateral Root Founder Cells to Inhibit
Root Initiation
W
Laurent Laplaze,a,1Eva Benkova,b,cIlda Casimiro,dLies Maes,b,cSteffen Vanneste,b,cRanjan Swarup,e
Dolf Weijers,f,2Vanessa Calvo,dBoris Parizot,b,c,gMaria Begon ˜a Herrera-Rodriguez,aRemko Offringa,f
Neil Graham,hPatrick Doumas,aJiri Friml,b,cDidier Bogusz,aTom Beeckman,b,cand Malcolm Bennette
aInstitut de Recherche pour le De ´veloppement, Unite ´ Mixte de Recherche Diversite ´ et Adaptation des Plantes Cultive ´es
(Agro.M/Institut National de la Recherche Agronomique/Institut de Recherche pour le De ´veloppement/Universite ´
Montpellier 2), Equipe Rhizogene `se, 34394 Montpellier cedex 5, France
bDepartment of Plant Systems Biology, Root Development Group, VIaams Instituut voor Biotechnologie, B-9052 Gent, Belgium
cDepartment of Molecular Genetics, Ghent University, B-9052 Gent, Belgium
dDepartmento de Ciencias Morfologicas y Biologia Celular y Animal, University of Extramadura, E-06071 Badajoz, Spain
eCentre for Plant Integrative Biology, School of Biosciences, University of Nottingham, Loughborough, LE12 5RD, United Kingdom
fInstitute of Biology, Leiden University, Wassenaarseweg 64 2333 AL Leiden, The Netherlands
gLaboratoire de Biologie du De ´veloppement des Plantes (Centre National de la Recherche Scientifique/Commissariat a ` l’Energie
Atomique/Aix-Marseille II), Commissariat a ` l’Energie Atomique Cadarache, 13108 St. Paul les Durance, France
hNottingham Arabidopsis Stock Centre, School of Biosciences, University of Nottingham, Loughborough, LE12 5RD, United
Kingdom
InArabidopsisthaliana,lateralrootsareformedfromrootpericyclecellsadjacenttothexylempoles.Lateralrootdevelopment
isregulatedantagonisticallybytheplanthormonesauxinandcytokinin.Whileagreatdealisknownabouthowauxinpromotes
lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to
disrupt lateral rootinitiation andthe regular patternofdivisions that characterizes lateral rootdevelopment in Arabidopsis.To
identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium
tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root
primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are
sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because
transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in
increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells
and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.
INTRODUCTION
The plant root system is made of a primary root that originates
during embryogenesis and lateral roots that form throughout the
life of the plant. Root architecture is influenced by numerous
environmental parameters. For example, the availability of nutri-
ents such as nitrate (Leyser and Fitter, 1998; Zhang and Forde,
2000), phosphate (Lopez-Bucio et al., 2002), and sulfate (Kutz
et al., 2002) has a strong effect on Arabidopsis thaliana lateral
root development (for review, see Lopez-Bucio et al., 2003;
Malamy, 2005). This plasticity of the root system is essential to
optimize nutrient acquisition in a heterogeneous and changing
environment and therefore represents an important agronomical
trait (Hodge, 2004).
Lateral roots originate from a small number of differentiated
cells situated at the periphery of the vascular tissues. In Arabi-
dopsis, lateral roots are derived from pericycle cells adjacent to
the xylem poles, called pericycle founder cells (Casimiro et al.,
2001; Dubrovsky et al., 2001). These cells undergo a defined
program of oriented cell divisions and expansion to form a lateral
root primordium (LRP; Malamy and Benfey, 1997; Dubrovsky
et al., 2001; Casimiro et al., 2003). The first step in lateral root
development (lateral root initiation) occurs in three adjacent
pericycle cell files. A polarized asymmetrical anticlinal division
takes place in two founder cells per cell file leading to the
formation of two short daughter cells surrounded by two larger
cells (stage I). Anticlinal divisions, cell expansion, and periclinal
divisions give rise to a simple four-layered LRP (stage IV). More
divisions and expansion result in the formation of a complex
stage VI LRP whose organization is similar to the primary root
1Address correspondence to laplaze@mpl.ird.fr.
2Current address: Laboratory of Biochemistry, Wageningen University
and Research Centre, Dreijenlaan 3, 6703 HA, Wageningen, The
Netherlands.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Laurent Laplaze
(laplaze@mpl.ird.fr).
WOnline version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.107.055863
The Plant Cell, Vol. 19: 3889–3900, December 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

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meristem. Cell expansion then drives the emergence of the LRP
fromtheparentroot(stageVIII)beforecelldivisionsresumeinthe
tip (lateral root meristem activation).
Auxin plays a central role during lateral root development
(reviewed in Casimiro et al., 2003). External auxin is required by
LRPs for initiation and development until they become self-
sufficient between stages III and V (Laskowski et al., 1995;
Himanen et al., 2002; Marchant et al., 2002; Casimiro et al.,
2003). It has been shown that for proper LRP development,
establishment of an auxin gradient with its maximum at the tip is
important (Benkova ´ et al., 2003). This gradient is dependent on
auxin transport mediated by PIN auxin efflux facilitators. Chem-
ical (inhibitors) orgenetic (mutants) interference with auxin trans-
port leads to defects in both auxin gradient establishment and
LRP development (Benkova ´ et al., 2003, Geldner et al., 2004).
In contrast with auxin, relatively little is known about the role of
cytokinins on lateral root development. Many reports describe
the inhibitory effect of cytokinins on lateral root formation
(Bo ¨ttgor, 1974; Goodwin and Morris, 1979; Wightman et al.,
1980), butthemechanism(s) ofcytokinin regulation isnotknown.
Arabidopsismutantsincytokininreceptors(Riefleretal.,2006)or
Arabidopsis response regulator genes (To et al., 2004; Mason
et al., 2005) involved in cytokinin signaling have root branching
phenotypes. Moreover, transgenic plants with reduced levels of
cytokinins due to the overexpression of genes encoding the
cytokinin-degrading enzyme cytokinin oxidase (CKX) exhibit
enhanced root growth and branching (Werner et al., 2001,
2003). We can conclude from these observations that the effect
of cytokinins is physiologically relevant and that endogenous
cytokinins act in vivo to inhibit lateral root development.
Itiscurrentlyunclearwhichstageoflateralrootdevelopmentis
inhibited by endogenous cytokinins. Mahonen et al. (2006) have
recently shown that cytokinin signaling is repressed in xylem-
pole pericycle cells, suggesting that lateral root founder cells
could bedeliberatelyshielded fromcytokininaction.Arabidopsis
CKX genes are expressed in LRPs (Werner et al., 2003), sug-
gesting that removal of the cytokinin signal is also important for
later stages of lateral root development (Schmu ¨lling, 2002).
Recently, exogenous cytokinin applications were shown to re-
press lateral root initiation in Arabidopsis (Li et al., 2006).
In this study, we report that lateral root founder cells are
sensitive to cytokinins, whereas young LRPs are not. We show
that cytokinins perturb the expression of PIN genesin lateral root
founder cells, preventing the formation of an auxin gradient that
is required to pattern LRP.
RESULTS
Cytokinins Inhibit Lateral Root Development
Cytokinins are known to be involved in multiple developmental
processes, including rhizogenesis (Haberer and Kieber, 2002).
To investigate the impact of elevating cytokinin levels during
lateralrootdevelopment,weinitiallyobservedtheconsequences
of growing Arabidopsis seedlings in the presence of various
concentrations of the cytokinins kinetin or 6-benzylaminopurine
(BAP). Root growth and lateral root density (number of emerged
lateral roots/cm primary root) were analyzed 10 d after germina-
tion. Kinetin concentrations of 0.1 and 0.5 mM had little effect on
primaryrootlength,reducinggrowth8.2and13.3%,respectively
(Figure 1A). A strong reduction of root growth was only observed
for kinetin concentrations $1 mM. By contrast, cytokinins
strongly reduced lateral root density at low concentration with
an average fourfold reduction for 0.1 mM kinetin compared with
nontreated plants (Figure 1B). BAP was found to be more active,
but the trend was the same (see Supplemental Figure 1 online).
Hence, lateral root development is more sensitive to cytokinin
treatment than primary root growth.
Cytokinins stimulate ethylene production in certain conditions
(Wang et al., 2002). To test whether the effect of elevated
cytokinin on lateral root development is mediated by ethylene,
we initially analyzed the effect of cytokinins in the presence of
aminoethoxyvinylglycine (AVG) (an inhibitor of ethylene biosyn-
thesis). AVG treatment prevented cytokinin inhibition of primary
root elongation consistent with previous reports (Cary et al.,
1995). However, the cytokinin-dependent inhibition of LRP initi-
ation and development was not rescued by AVG (see Supple-
mental Figure 2 online), suggesting that the cytokinin effect on
LRP formation is independent of ethylene biosynthesis. We
further addressed the relation between ethylene and cytokinin
inLRPformationbyanalyzingtheeffectofcytokininontherootof
the ethylene-insensitive mutant etr1, which is defective in ethyl-
ene perception (Chang et al., 1993). Cytokinins had no signifi-
cant effect on etr1 primary root growth (Figure 1C). By contrast,
the ethylene-insensitive mutant, like control plants, showed a
significant reduction in lateral root density in the presence of
cytokinins (Figure 1D). Hence, results obtained using an ethylene-
insensitive mutant and an inhibitor of ethylene biosynthesis
indicate that cytokinins exert their effects on lateral root de-
velopment independently of ethylene. However, the inhibition
of primary root growth by cytokinins appears to be ethylene
dependent.
Exogenously Applied Cytokinins Perturb Both Initiation
and Organization of LRPs
To determine at which stage of lateral root development cyto-
kininsaredisrupted,seedlingrootswereclearedandthenumber
and developmental stages of LRPs were analyzed (Figure 2).
Ten-day-oldseedlingstreatedwithcytokininsshowedareduced
number of LRPs compared with plants grown without cytokinins
(Figure 2A), therefore indicating that cytokinins disrupt lateral
root initiation. This was confirmed using an earlier described
lateral root inducible system (Himanen et al., 2002). Seedlings
are germinated in the presence of the polar auxin transport
inhibitor naphthylphthalamic acid (NPA), which blocks lateral
rootinitiation(Casimiroetal.,2001).Rootsarethentransferredto
auxin-containing media, and in a period of 12 h, xylem pole cells
throughout the entire root pericycle initiate their first anticlinal
divisions (Himanen et al., 2002). The use of transgenic plants
containing a promoter:b-glucuronidase (GUS) fusion for the
CYCB1;1 gene in this lateral root inducible system makes it
possible to time precisely the very first divisions in the pericycle.
SeedsoftheProCYCB1;1:GUStransgeniclineweregrownfor72
h in the presence 10 mM NPA. Seedlings were then transferred
to growth medium containing 10 mM 1-naphtalene acetic acid
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(NAA) or 10 mM NAA supplemented with 0.1, 1, or 10 mM BAP,
respectively. Samples were tested for GUS activity by histo-
chemical staining at 2-h intervals after transfer. NAA treatment
was found to induce pericycle cell divisions from 6 h onwards
starting near the root apical meristem and gradually moving
basipetally as described earlier (Himanen et al., 2002). At 12 h,
the complete pericycle at the protoxylem poles was activated
(Figure 2C). In the presence of BAP, this activation was delayed
and the first cell divisions could be detected only after 8 h of
incubation on both hormones. At 12 h, only the most apical part
of the pericycle appeared to be induced (Figure 2D). Therefore,
cytokinin treatments clearly delayed cell divisions in the pericy-
cle. This effect was found to be dose dependent. When plants
were grown for 48 h in the presence of 10 mM NPA and then
transferred to medium containing both 10 mM NPA and 10 mM
BAP for another 24 h before activation in the presence of
cytokinins (10 mM NAA þ 10 mM BAP), a more pronounced
delay in lateral root initiation was observed. The first cell cycle
activity could be detected in the pericycle only at 12 h after
treatment (Figure 2E). After 24 h, the pericycle was activated
>50% of the primary root length (Figure 2F), and it took another
24 h to induce the entire pericycle. Hence, cytokinins perturb the
first anticlinal division leading to lateral root development. This
result is in agreement with previous studies (Li et al., 2006).
Theeffectofcytokininsonlateralrootinitiationdoesnotentirely
explain the reduced lateral root number in the cytokinin-treated
plants(Figures1Band2A).Wealsoobservedthatthedistribution
of developmental stages of primordia was altered by cytokinins,
with an increased proportion of stages IV and V and a decreased
proportion of later stages compared with plants grown without
cytokinins (Figure 2B). This suggests that the development of
some LRPs was either delayed or stopped around stages IV and
V and would, together with reduced lateral root initiation, explain
the reduction in the number of emerged lateral roots in cytokinin-
treated plants. Detailed morphological studies revealed that
cytokinin-treated plants showed abnormal patterns of cell divi-
sions in a significant proportion of LRPs (53.7% for 0.1 mM
kinetin, n ¼ 298 LRP from 12 roots) that were rarely observed in
control plants(2.04%,n¼442LRPfrom12roots).Insomestage
I primordia, cytokinin treatment was observed to cause tangen-
tial and oblique divisions (Figure 2H) in place of the normal
pattern of anticlinal divisions (Figure 2G). In stage II primordia,
Figure 1. Cytokinins Block Lateral Root Development Independently of Ethylene.
(A) Cytokinins reduce primary root growth.
(B) Cytokinins reduce lateral root density.
(C) The etr1 mutant is insensitive to cytokinin effect on primary root growth.
(D) The etr1 mutant is sensitive to cytokinin-mediated inhibition of lateral root development.
(A) and (B) Plants were grown on vertical agar plates (half-strength Murashige and Skoog [MS] and 1.2% phytagel) supplemented with 0 (control, n ¼
50), 0.1 (n ¼ 45), and 0.5 mM (n ¼ 45) kinetin. Root length and the number of emerged lateral roots were measured 10 d after germination (DAG).
(C) and (D) Wild-type (Col-0) and etr1-1 plants were grown for 10 d on vertical agar plates containing no (MS) or 0.5 mM kinetin (MSþkinetin). Root length
and the number of emerged lateral roots were recorded using a stereomicroscope; n ¼ 37 (Col-0, MS), 39 (Col-0, MSþkinetin), 27 (etr1-1, MS), and 34
(etr1-1, MSþkinetin).
The values shown are means 6 SD. Significance was analyzed by analysis of variance (ANOVA) test. * P < 0.05 compared with untreated ([A] and [B]) or
wild-type ([C] and [D]) plants. LR, lateral roots.
Cytokinins and Lateral Root Development3891

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Figure 2. Cytokinin Treatment Perturbs Lateral Root Initiation and Patterning.
(A) Cytokinins affect lateral root initiation.
(B) Cytokinins alter the stage distribution of LRPs.
(C) Cell divisions occur along the entire xylem-pole pericycle 12 h after induction.
(D) Cytokinin treatment perturbs the induction of cell divisions in the pericycle 12 h after induction.
(E) Pretreatment with 10?5M BAP leads to a strong inhibition of cell divisions in the root pericycle 12 h after induction.
(F) Pretreatment with 10?5M BAP leads to a strong inhibition of cell divisions in the root pericycle 24 h after induction.
(G) Stage I LRPs exhibit a regular pattern of anticlinal divisions (arrowheads). X, xylem.
(H) Cytokinin treatment causes abnormal tangential and oblique divisions (arrows).
(I) In stage II primordia, anticlinal divisions only occur in the outer layer (OL).
(J) Cytokinin treatment causes ectopic anticlinal divisions within the inner layer (IL).
(K) Stage III/IV LRPs form a dome shape due to rounds of periclinal divisions in the central cells of the outer layer.
(L) Cytokinin treatment disrupts the regular pattern of periclinal divisions in central cells of the outer layer, causing the developing LRP to appear
flattened. Instead, each cell in the outer layer underwent an additional round of anticlinal division giving rise to double the normal number of cells (see
numbered cells).
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anticlinal divisions normally only occur in the outer layer (Figure
2I), but cytokinin treatment caused ectopic anticlinal divisions in
cells within the inner layer (Figure 2J). Stage III/IV LRPs normally
form a dome shape due to rounds of periclinal divisions in the
central cells of the outer layer (Figure 2K). However, following
cytokinin treatment, central cells in the outer layer failed to
undergo periclinal divisions, causing the developing LRP to
appear flattened (Figure 2L). Instead, each cell in the outer layer
underwent an additional round of anticlinal division giving rise to
double the normal number of cells (Figure 2L). Central cells of
stage VI LRP normally undergo another round of periclinal
division (Figure 2M) to give rise to columella tissues (Malamy
and Benfey, 1997). However, high levels of cytokinin disrupt this
round of periclinal divisions (Figure 2N). As a result, the mor-
phology of someofthe cytokinin-treated LRPthatemerge(stage
VIII)appearsdisorganizedparticularlyattheapex(Figure2P).We
conclude from our observations that cytokinin treatment results
in a disorganized pattern of divisions in developing LRP leading
to perturbed LRP organization.
Taken together, these results show that increasing exogenous
cytokinin concentration leads to reduced lateral root initiation
and disorganization of some LRPs.
Direct Effect of Cytokinins on Lateral Root Founder Cells
TopreciselydelimitwhichstageofLRdevelopmentwassensitive
to the cytokinin signal, we ectopically expressed the Agrobacte-
rium tumefaciens cytokinin biosynthesis enzyme isopentenyl-
transferase (IPT; Akiyoshi et al., 1984) in either xylem-pole
pericycle cells or young LRPs. This was achieved by targeting
the expression of anupstream activation sequence (UAS)–linked
IPTtransgeneusingthexylem-polepericyclecellorLRP-specific
GAL4-GFP enhancer trap lines J0121 and J0192, respectively
(Laplazeetal.,2005).Sincedifferentreportshavesuggestedthat
cytokininsactwheretheyareproduced(Heweltetal.,1994;Faiss
et al., 1997; Nordstro ¨m et al., 2004), we assume that the pheno-
typic effects of IPT misexpression are due to a local effect of
cytokinins. However, we cannot rule out translocation of cytoki-
ninstoothersitesofaction.TransgenicplantsharboringUAS-IPT
did not show any phenotype in the absence of GAL4. However,
when the UAS-IPT line was crossed with another transgenic line
expressing GAL4 fused to the L1-specific LTP1 promoter (Weijers
et al., 2003), LTP1?IPT F1 plants showed reduced root and
hypocotyl growth, pale cotyledons, and serrated leaves (see
Supplemental Figure 3 online). This phenotype is very similar
cytokinin-overproducing plants (Rupp et al., 1999) or wild-type
plants sprayed with cytokinins, thus confirming the functionality
of the UAS-IPT transgene.
Homozygous J0121 or J0192 plants were crossed with ho-
mozygous UAS-IPT plants. F1 J0121?IPT and J0192?IPT
plants were grown on vertical plates together with control plants
(J0121 3 Col-0 and J0192 3 Col-0), and root length and lateral
root number were measured 10 DAG. J0121?IPT seedlings had
shorter roots than control plants (Figure 3A). No emerged lateral
roots were observed on 10-d-old J0121?IPT plants (n ¼ 30)
compared with an average of 4.38 6 2.78 lateral roots for control
(J0121 3 Col-0) plants (n ¼ 33). Roots were cleared and the
number and stages of LRPs were recorded. J0121?IPT plants
were able to develop LRPs but had a LRP density reduced by
42% (Figure 3B). This indicates that targeted cytokinin biosyn-
thesis in the xylem-pole pericycle cells disrupts lateral root
initiation. This was not due to a change in pericycle cell spec-
ification since GFP expression was not changed in J0121?IPT
compared with control J0121 3 Col-0 plants (see Supplemental
Figure 4 online). By contrast, J0192?IPT seedlings did not show
any significant change in root length or lateral root density
comparedwithcontrol(J01923Col-0)plants(seeSupplemental
Figure 5 online). Therefore, localized cytokinin biosynthesis in
newly initiated primordia has no effect on lateral root develop-
ment. Our study has revealed stage- and cell-specific effects of
cytokininsapplication.Xylem-polepericyclecellsaresensitiveto
ectopic cytokinins biosynthesis, whereas young LRPs (stages I
to IV) are not.
Surprisingly, in 10-d-old J0121?IPT seedlings, LRPs that did
form had not developed beyond stage V (Figure 3C). Closer
inspection of LRP morphology revealed that the cellular organi-
zation of LRP in J0121?IPT plants appeared disorganized
compared with the wild type (see Supplemental Figure 6 online).
We conclude that exposing xylem-pole pericycle cells to cyto-
kinin disrupts an important patterning process, which later
impacts the cellular organization of the developing LRP.
We tested whether the effect of cytokinins on lateral root
founder cells was physiologically significant by lowering the
endogenous level of cytokinins specifically in those cells.
We targeted the expression of UAS-linked Arabidopsis cytoki-
nin degrading enzyme cytokinine oxidase 1 (UAS-CKX1; Ioio
et al., 2007) in xylem-pole pericycle cells using J0121. F1
J0121?CKX1 plants were grown on vertical plates together with
control (J0121 3 Col-0) plants, and root length and lateral root
number were recorded 11 DAG. CKX1 expression in xylem-pole
Figure 2. (continued).
(M) Central cells in stage VI LRPs undergo another round of periclinal division (arrows).
(N) Cytokinins disrupt this round of periclinal divisions.
(O) Emerged lateral root with a sharp apex due to the formation of the central columella.
(P) Some cytokinin-treated LRPs that emerge appear disorganized particularly at the apex
(A) and (B) Ten-day-old plants grown on vertical agar plates containing 0 (control, n ¼ 12), 0.1 (n ¼ 14), and 0.5 mM (n ¼ 10) kinetin were cleared, and the
number and stages (Malamy and Benfey, 1997) of LRPs were recorded.
(C) to (F) ProCYCB1;1:GUS seeds were germinated and grown as described (Himanen et al., 2002). Bars ¼ 500 mm.
(G) to (P) Ten-day-old plants grown on vertical agar plates containing 0, 0.1, and 0.5 mM kinetin (n ¼ 10/condition) were cleared and mounted according
to Malamy and Benfey (1997). Bars ¼ 25 mm.
Cytokinins and Lateral Root Development3893

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pericycle cells had no significant effect on root growth (Figure
3D).LateralrootdensitywassignificantlyhigherinJ0121?CKX1
plants comparedwithcontrol plants(Figure3E). Hence,lowering
the endogenous cytokinin concentration in lateral root founder
cells results in increased lateral root formation. We therefore
concludethatcytokininsareimportantendogenousregulatorsof
lateral root initiation and that the effects we observed in our IPT
transactivation experiments (gain of function) are physiologically
significant.
Auxin-Mediated Activation of Cytokinin-Accumulating
Pericycle Cells Does Not Rescue Lateral Root Formation
Given the importance of auxin to lateral root organization
(Benkova ´ et al., 2003), we tested whether exogenous auxin could
rescue the lateral root defect of plants producing cytokinin in the
xylem-pole pericycle cells. J0121?IPT and control (J0121 3
Col-0) plants were grown for 5 d on vertical plates and then
transferred to vertical plates containing different concentrations
(0, 0.1, 1, and 10 mM) of the naturally occurring indole-3-acetic
acid(IAA).Theeffectsofdifferentauxins(IAA,syntheticNAA,and
2,4-D) were also tested at 1 mM.
In contrast with control plants, none of the auxin treatments
were able to induce the formation of fully developed lateral roots
in J0121?IPT plants (Figures 4A to 4C). By contrast, control
plants treated with either IAA or NAA showed more lateral roots
and LRPs (Figures 4A and 4B). The most dramatic phenotype
was obtained with 2,4-D (Figure 4C) and was analyzed more
carefully. Treatment with 1 mM 2,4-D activates cell divisions in all
xylem-pole pericycle cells, leading to lateral root formation along
the entire length of the primary root (Himanen et al., 2002).
Treatment with 1 mM 2,4-D completely blocked primary root
growth of control and J0121?IPT plants (Figure 4D) and also
induced pericycle cells division in both control and J0121?IPT
plants,demonstratingthatJ0121?IPTplantsarestillsensitiveto
auxin. Five days after transfer to 2,4-D, LRPs were present along
Figure 3. Targeting Cytokinin Biosynthesis or Degradation in Lateral
Root Founder Cells Disrupts Lateral Root Formation.
(A) J0121?IPT (n ¼ 30) plants have a reduced root length compared with
control (J0121 3 Col-0) plants (n ¼ 33). The values shown are means 6
SD. Significance was analyzed by ANOVA test. * P < 0.05 compared with
control plants.
(B) J0121?IPT plants (n ¼ 12) show a reduced LRP density com-
pared with control (J0121 3 Col-0) plants (n ¼ 10). The values shown are
means 6 SD. Significance was analyzed by ANOVA test. * P < 0.05 com-
pared with control plants.
(C) Ten-day-old J0121?IPT plant (n ¼ 12) LRP distribution. No LRPs
beyond stage V were observed.
(D) J0121?CKX1 (n ¼ 22) plants show no significant change in root
length compared with control (J0121 3 Col-0) plants (n ¼ 25). The values
shown are means 6 SD. Significance was analyzed by ANOVA test.
(E) J0121?CKX1 plants (n ¼ 22) show a increase lateral root density
compared with control (J0121 3 Col-0) plants (n ¼ 25). The values shown
are means 6 SD. Significance was analyzed by ANOVA test. * P < 0.05
compared with control plants.
Plants were grown on vertical agar plates (half-strength MS and 1.2%
phytagel). Ten-day-old J121?IPT and control plants ([B] and [C]) and
11-d-old J121?CKX1 and control plants ([D] and [E]) grown on vertical
agar plates were cleared, and the number and stages (Malamy and
Benfey, 1997) of LRPs were recorded.
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the entire length of the control (J0121 3 Col-0) root in front of the
xylem pole(Figure 4E). Bycontrast,J0121?IPT plantsshowed a
continuous layer of cells three to four cells wide in front of the
xylem poles but no discrete primordia (Figure 4F). In this system,
J0121?IPTplantswerenotabletodevelopLRPevenafterbeing
exposed to 2,4-D for 1 week. Thus, auxins do not rescue the
lateral root initiation defect of J0121?IPT plants. This is consis-
tent with previous results indicating that auxin cannot rescue the
cytokinin-mediated inhibition of lateral root initiation (Li et al.,
2006). Our results further suggest that cytokinin accumulation in
pericycle cellsdoes not prevent the auxin-mediated activation of
cell divisions but blocks the developmental program of lateral
root initiation.
Cytokinins Disrupt PIN-Dependent Formation of an Auxin
Maximum during Lateral Root Development
We first tested whether cytokinins inhibit lateral root initiation by
changing the sensitivity of lateral root founder cells to auxin by
monitoring the expression of the auxin-sensitive ProDR5:GUS
marker in the lateral root inducible system. Seeds of the
ProDR5:GUS transgenic linewere grown for72 hin thepresence
10 mM NPA. Seedlings were then transferred to growth medium
containing 10?5M NAA or 10 mM NAA supplemented either with
0.1,1,or10mMBAP,respectively.SamplesweretestedforGUS
activity by histochemical staining after transfer. We observed
that the auxin-responsive promoter ProDR5:GUS was activated
at the same time in the lateral root induction system in the
presence or absence of cytokinins (see Supplemental Figure 7
online), therefore indicating that cytokinins did not perturb auxin
perception in xylem-pole pericycle cells.
Benkova ´ et al. (2003) have shown that a localized auxin
maximum in newly developed LRP influences the patterning of
the emergent organ. To address whether cytokinins affect the
patterning of LRP by modulating auxin distribution, we tested its
effect on the spatial expression of the auxin-responsive reporter
ProDR5:GUS. In control plants, ProDR5:GUS expression was
detected in the pericycle in presumptive LRP founder cells and
after the formation of short initials by anticlinal division in these
cells (Figure 5A). During progression to the later stages, a gra-
dient of GUS activity with a maximum at the tip was gradually
established (Benkova ´ et al., 2003; Figure 5C). However, expres-
sionoftheProDR5:GUSreporterinseedlingsgrownoncytokinins
showed a strikingly different pattern. ProDR5:GUS signal was
occasionally detected along the root vasculature or sporadically
in pericycle cells (Figure 5B). In contrast with control seedlings,
this auxin response was only rarely accompanied by the anticli-
nal division leading to LRP initiation. ProDR5:GUS signal in LRP
of the seedlings germinated on cytokinin was weaker and more
diffuse, and the maximum at the primordia tip was often missing
(68%) compared with the untreated control (23%; Figure 5D).
Also, a larger portion of cytokinin-grown LRP did not show any
staining (cytokinin 60%, control 40%; n ¼ 48 and 44), indicating
lower auxin status. Hence, cytokinins appear to perturb the
formation of an auxin maximum in LRP from the first division on.
Formation of an auxin maximum in LRP has been shown to
depend ontheactivityofPINauxinefflux carriers(Benkova ´ etal.,
2003).WethereforeexaminedtheeffectofcytokininsonPINgene
expressionduringlateralrootdevelopment.Tomonitortheeffect
of cytokinins on PIN gene expression during the initial phase of
lateral root development, we made use of the lateral root induc-
ible system (Himanen et al., 2002). The mRNA abundance of
PIN1, 2, 3, 4, 6, and 7 was analyzed 6 h after transfer to the
induction medium with or without addition of cytokinins. PIN1, 2,
3, and 7 were strongly induced 6 h after transfer on the induction
medium.ThisPINgeneinductionwasreducedinthepresenceof
Figure 4. Auxins Do Not Rescue the J0121?IPT Lateral Root Phenotype.
Plants were grown for 5 d on vertical plates and then transferred on new vertical plates containing 0 or 1 mM auxin (IAA, NAA, or 2,4-D). Root length and
lateral root density were analyzed 0, 2, and 5 d after transfer. Bars ¼ 5 mm in (A) to (C) and 100 mm in (E) and (F).
(A) Control (J0121 3 Col-0, left) and J0121?IPT (right) plants 5 d after transfer on vertical plates containing IAA.
(B) Control (J0121 3 Col-0, left) and J0121?IPT (right) plants 5 d after transfer on vertical plates containing NAA.
(C) Control (J0121 3 Col-0, left) and J0121?IPT (right) plants 5 d after transfer on vertical plates containing 2,4-D.
(D) J0121?IPT plants are still sensitive to auxin-mediated inhibition of root growth. Transfer of control and J0121?IPT plants on auxin-containing
medium 5 DAG (arrow) leads to an arrest of root growth; n ¼ 19 (control), 17 (control; 2,4-D), 20 (J0121?IPT), and 20 (J0121?IPT; 2,4-D).
(E) View of LRPs on a control (J0121 3 Col-0) plant 5 d after transfer on 2,4-D–containing medium.
(F) View of the thickening of the pericycle in J0121?IPT plants 5 d after transfer on 2,4-D–containing plates.
Cytokinins and Lateral Root Development 3895

Page 8

cytokinins (Figure6).Theseresultsshowthatcytokinintreatment
impacts PIN genes expression early during lateral root initiation.
Cytokinin-induced changes in PIN gene expression were also
examined during later stages of lateral root development using
transcriptional and translational reporter fusions. Marker lines
were grown on medium with or without cytokinins, and their
expression pattern was recorded. PIN1 expression in LRPs was
broader, and the boundary between inner and outer layers was
less clear compared with controls, where PIN1 expression is
restricted to derivatives of inner layer (Figures 5F and 5H versus
5E and 5G; Benkova ´ et al., 2003; cytokinin, 53% LRP; control,
20% LRP; n ¼ 28 and 35). Cytokinin also affected the spatial
expressionofotherPINgenes.Forexample,analysisofProPIN6:
GUS lines revealed more diffuse expression in LRPs (Figures 5J
and 5L versus 5I and 5K; cytokinin, 68%; control, 27%; n ¼ 25
and40).CytokininsthereforeappeartoperturbthepatternofPIN
genes expression in LRP. Expression of PIN1, 2, 3, 4, and 7 was
also strongly downregulated in the shoot of cytokinin-treated
plants (see Supplemental Figure 8 online). As LRP initiation is
supposed to be dependent on auxin transported from the shoot
(Reedetal.,1998),thisgeneraldownregulationofPINexpression
might be one of the causes for reduction in LRP initiation in
cytokinin-grown seedlings. By contrast, cytokinin treatment had
no effect on the expression of the auxin influx carrier AUX1
(Figure 5N versus 5M) as marked by a ProAUX1:AUX1-YFP
translational reporter fusion (Swarup et al., 2004). In conclusion,
our results suggest that cytokinins inhibit lateral root initiation by
interfering either directly or indirectly with PIN-dependent auxin
distribution.
Cytokinins disrupt the formation of the auxin maximum, which
patterns LRP. However, the absence of discrete LRP in 2,4-D–
treated J121?IPT plants suggests that cytokinins also cause
defects in establishment of primordia margins. This possibility is
consistent with the observed changes in PIN6 expression in LRP
(Figures 5J to 5L). Moreover, another margin marker, CUC3
(Vroemen et al., 2003), was not expressed in LRP developing on
cytokinins in contrast with controls (Figure 5P versus 5O). This
altered pattern of PIN6 and CUC3 expression in LRP is likely to
reflect defects in establishment of primordia margins during
lateral root initiation.
DISCUSSION
Cytokinins Regulate Lateral Root Development in a
Stage-Specific Manner
Auxin is considered the major regulator of lateral root develop-
ment based on a large body of physiological, genetic, and
Figure 5. Cytokinin Treatment Affects ProDR5:GUS, PIN, and CUC3 Expression.
(A) ProDR5:GUS is expressed in dividing pericycle cells of control roots.
(B) ProDR5:GUS is occasionally expressed in pericycle cells of cytokinin-grown roots but is not accompanied by initiation of LRP development.
(C) Establishment of the ProDR5:GUS gradient in LRPs of control roots.
(D) The ProDR5:GUS gradient is not established in LRPs in cytokinin-treated roots.
(E) ProPIN1:PIN1-GFP is associated with the central zone of LRP in control roots.
(F) ProPIN1:PIN1-GFP expression is more diffuse in LRPs of cytokinin-grown roots.
(G) ProPIN1:GUS is expressed at the base and center of LRP in control roots.
(H) ProPIN1:GUS is expressed at the base of LRP of cytokinin-grown roots.
(I) ProPIN6:GUS is expressed at the margins of stage IV LRPs in control roots.
(J) ProPIN6:GUS expression is not restricted to the margins of LRPs grown on cytokinins.
(K) ProPIN6:GUS is expressed at the base and margins of stage VI LRPs in control roots.
(L) ProPIN6:GUS is broadly expressed in LRPs grown on cytokinins.
(M) ProAUX1:AUX1-YFP is expressed throughout stage V LRPs of control roots.
(N) ProAUX1:AUX1-YFP expression in unaffected by cytokinin treatment.
(O) ProCUC3:GUS is expressed at the margins of stage VII LRPs.
(P) ProCUC3:GUS is not expressed in stage V LRPs grown on cytokinins.
Marker lines were grown for 10 d on MS (top row) or on MS supplemented with 0.5 mM kinetin (bottom row). Bars ¼ 25 mm.
3896 The Plant Cell

Page 9

molecular cell biological evidence (reviewed in Casimiro et al.,
2003).Here,wedemonstratethatcytokininsalsoinfluencelateral
root development. This is in agreement with previous results
showing that transgenic plants with reduced cytokinin content
duetotheconstitutiveexpressionofageneencodingacytokinin-
degradingenzyme,CKX,displayincreasedrootbranching(Werner
et al., 2001, 2003). Similarly, Arabidopsis cytokinin receptor mu-
tants show increased lateral root formation (Riefler et al., 2006).
Taken together, these results indicatethatcytokinins act invivoto
regulate root system architecture.
Our study has demonstrated that cytokinins influence lateral
root formation independently of ethylene at a very early devel-
opmental stage. In Arabidopsis, lateral root founder cells are
derived from three axial files of root pericycle cells that are
adjacent to the two xylem poles (Dubrovsky et al., 2001). A
previous study showed that exogenous applications of cytoki-
nins inhibit lateral root initiation in those cells (Li et al., 2006).
However, the main disadvantage of this experimental approach
(exogenous application of plant hormones) is that it does not
differentiate direct and indirect effects. We used a GAL4-based
transactivation strategy to overcome this problem. This enabled
ustoshowthatlateralrootfoundercellsaresensitivetocytokinin
application, while LRPs are not. Moreover, we found that the
endogenous level of cytokinins in lateral root founder cells limits
lateral root formation. Our data indicate that cytokinins disrupt
lateral root initiation directly in xylem pole pericycle cells in
planta.
Cytokinins have recently been demonstrated to influence cell
differentiation in Arabidopsis root apical meristem (Mahonen
et al., 2006; Ioio et al., 2007). While cytokinins have been shown
to repress protoxylem cell specification (Mahonen et al., 2006),
these phytohormones do not appear to block xylem pole peri-
cycle cell fate, only slightly delaying the onset of expression of
the J0121 marker close to the root apical meristem. Hence,
targeted expression of IPT in xylem-pole pericycle cells does not
disrupt primordium formation by altering lateral root founder cell
fate. Instead, our data suggest that cytokinins disrupt a later
developmental event involving the polarized asymmetric cell
division in two adjacent founder cells that normally leads to the
formation of two short daughter cells surrounded by two larger
daughter cells. Detailed morphological analyses of cytokinin-
treated roots revealed an abnormal pattern of cell divisions from
the earliest developmental stage onwards. For example, in stage
I primordia, tangential and oblique divisions were observed in
place of the normal pattern of anticlinal divisions. Similarly, in
stage II primordia, anticlinal divisions normally only occur in the
outer layer but cytokinin treatment caused ectopic anticlinal
divisions in cells within the inner layer. We conclude that cyto-
kinins disrupt the organization and development of LRPs.
Cytokinins Disrupt PIN-Dependent Lateral Root Initiation
Howcouldexposinglateralrootfoundercellstocytokinindisrupt
the subsequent organization and development of LRPs? Auxinis
known to trigger the initial asymmetric cell division (Vanneste
Figure 6. PINmRNAAbundanceIsReducedbyCytokininsduringLateral
Root Initiation.
Plants were grown in the lateral root–inducible system. RNAs were
extracted from differentiated parts of roots 0 (control) and 6 h after
induction in the presence of 0 or 10 mM BAP. Expression levels were
normalized to ACTIN2 and are indicated as percentages of the expres-
sion in control plants. Presented values are means 6 SD.
Figure 7. Model of Cytokinin–Auxin Interaction during Root Develop-
ment.
(A) Lateral root initiation is triggered by auxin perception by some xylem-
pole pericycle cells leading to an asymmetric cell division. Cytokinins do
not block auxin perception or response in lateral root founder cells but
act downstream to perturb the asymmetric cell division.
(B) Lateral root initiation requires auxin-induced cell fate respecification
and cell cycle progression (Vanneste et al., 2005). Cell fate respecifica-
tion depends on the expression of PIN genes to create an auxin gradient
responsible for asymmetric cell division and acquisition of LRP identity.
Cytokinins inhibit this step by downregulating PIN gene expression.
Cytokinins and Lateral Root Development3897

Page 10

et al., 2005; Figure 7A). However, we have demonstrated in this
study that cytokinins did not change the perception of auxin in
lateral root founder cells (Figure 7A). Instead, we observed that
auxin was still able to induce cell divisions in xylem-pole pericy-
cle cells expressing the IPT gene but that this led to disorganized
primordia. Interestingly, this phenotype is reminiscent of plants
perturbed in PIN-mediated polar auxin transport (Benkova ´ et al.,
2003; Geldner et al., 2004). The organization of LRP is known to
be dependent on the coordinated expression of multiple PIN
genes to create an auxin maximum (Benkova ´ et al., 2003). We
observed that cytokinin treatment disrupted the induction of
some PIN genes during lateral root initiation (i.e., the very first
division of lateral root founder cells). Moreover, the early estab-
lishment of an auxin gradient was perturbed by cytokinins. This
suggests that cytokinins interfere with the initial asymmetric
division, and the resulting perturbation could explain the subse-
quent changes in the pattern of cell division and cell fate in
cytokinin-treated plants as revealed by altered expression of
PIN6 or CUC3 markers that define the LRP flanks.
In conclusion, we propose the following model to explain
cytokinin–auxin interaction during lateral root initiation (Figure
7B). Lateral root initiation requires the coordinated action of cell
cycle progression and cell fate respecification in lateral root
founder cells both induced by auxin (Vanneste et al., 2005).
These two processes can be uncoupled by mutation in the
Diageotropica gene in tomato (Solanum lycopersicum) that re-
sults in proliferative cell divisions but no lateral root initiation in
the pericycle in reponse to auxin (Ivanchenko et al., 2006).
Similarly, overexpression of a D-type cyclin (CYCD3;1) in the
Arabidopsis solitary root/iaa14 mutant leads to proliferative cell
divisions but is not sufficient to rescue lateral root initiation
(Vanneste et al., 2005). Thus, cell cycle activation in xylem-pole
pericycle cells is not sufficient for lateral root initiation. It also
requires auxin to induce the expression of PIN genes during
lateralrootinitiation(Vannesteetal.,2005;Vietenetal.,2005;this
study)tocreateanauxingradientresponsibleforasymmetriccell
divisionleadingtocellfaterespecificationandacquisitionofLRP
identity as shown by the phenotype of Arabidopsis plants
perturbed in polar auxin transport (Benkova ´ et al., 2003; Geldner
et al., 2004). Our results indicate that cytokinins do not block
lateral root initiation in lateral root founder cells by acting on
auxin-induced cell division but rather byinhibiting auxin-induced
cell fate respecification by downregulating PIN gene expression.
The endogenous source of cytokinins responsible for lateral root
development inhibition in planta remains to be defined. Cytoki-
nins are produced in a wide range of tissues and organs.
Interestingly, the root cap has been proposed as a site of
cytokinin production (Miyawaki et al., 2004) and genetic ablation
of the root cap stimulate lateral root initiation (Tsugeki and
Fedoroff, 1999).Future work willfocus on the identification of the
source of cytokinins that control lateral root initiation and how
cytokinin production is regulated by environmental factors, such
as nutrient concentrations.
Cytokinins and auxin have antagonistic effects on many as-
pects of plant development, including lateral root formation. It
was shown that auxin can directly downregulate cytokinin bio-
synthesis, while cytokinins had little effect on auxin biosynthesis
(Nordstro ¨m et al., 2004). Our results suggest that cytokinins act
on auxin homeostasis by changing auxin transport (via the
downregulation of PIN gene expression) rather than changing
auxin biosynthesis or perception. This illustrates that a complex
network of interacting hormones, including auxin and cytokinins,
regulates root architecture. An exciting challenge in the years to
come will be to understand this network of interacting hormonal
signals that controls lateral root formation.
METHODS
Plant Lines and Growth Conditions
C24, Col-0, and etr1-1 seeds were obtained from the Nottingham
Arabidopsis Stock Centre (http://nasc.nott.ac.uk/).
J0121 (C24) belongs to a collection of GAL4-GFP enhancer trap lines
available through the stock centers (http://www.plantsci.cam.ac.uk/
Haseloff). J0192 (C24) was isolated from a collection of 401 GAL4-GFP
enhancer trap lines generated by root transformation of C24 wild-type
plants during a screen for lateral root expressed lines (Laplaze et al.,
2005).
The ProUAS:IPT construct was created by cloning the Agrobacterium
tumefaciens IPT coding sequence (Akiyoshi et al., 1984) between the
6xUAS promoter and the NOS terminator in pSDM7022 (Weijers et al.,
2003) and subsequent transfer of the ProUAS:IPT:tNOS gene into
pSDM7006 (Weijers et al., 2003). The construct was transformed into
Col-0 wild-type plants, and transgenic lines were selected based on
T-DNA number, wild-type phenotype, and GAL4-dependent GUS ex-
pression as described (Weijers et al., 2003). The ProUAS-CKX1 trans-
genic line was described previously (Ioio et al., 2007)
Lines carrying ProPIN1:GUS, ProPIN1:PIN1-GFP, ProPIN3:GUS,
ProPIN6:GUS, ProDR5:GUS, and ProCUC3:GUS for analysis of expres-
sion in LRPs were described previously (Benkova ´ et al., 2003).
Plants were grown at 238C, 60% humidity, in 60 mE constant light on
verticalhalf-strengthMS1.2%phytagelplatesunderlong-dayconditions
(16 hlight/8 hdark). Seedswere surface sterilized and cold treated for2d
at 48C in the dark before transfer to the growth chamber. Plants in soil
were grown in a 1:1 (v/v) compost/vermiculite mix in a growth room at
218C in a 16-h-light/8-h-dark cycle.
Lateral root induction in the whole pericycle was performed according
to Himanen et al. (2002).
Root length was measured from digital images of the plates using the
NIH Image 1.62 software. Emerged lateral roots were counted using a
binocular. Data were analyzed using the Excel statistical package.
Experiments were repeated at least two times independently.
Microscopy
GUS activity was assayed by immersing seedlings in a staining solution
(Svistoonoff et al., 2003) at 378C. To limit the diffusionof the blue staining,
5 mM K3Fe(CN)6and K4Fe(CN)6were added. Tissues were cleared in
70% ethanol for 2 d. Tissues were then immersed in 50% (v/v) ethanol/
10%(v/v)glycerolfor2h,30%(v/v)ethanol/30%(v/v)glycerolfor2h,and
in 50% (v/v) glycerol for 2 h. Seedlings were then mounted in 50% (v/v)
glycerol and visualized on a DMRB microscope (Leica).
Quantitative RT-PCR
Col-0 seeds were germinated on medium containing 10 mM NPA and
transferred 3 d after germination under continuous light to 10 mM NAA or
10 mM NAA þ 10 mM BAP for 6 h. The root apical meristems were cut off,
and the shoots were removed by cutting below the adventitious root
primordia. Only the differentiated part of the root was used for RNA
3898 The Plant Cell

Page 11

extraction using the RNeasy kit (Qiagen). Poly(dT) cDNA was prepared
from 1 mg of total RNA with Superscript III reverse transcriptase
(Invitrogen) and quantified on an I cycler apparatus (Bio-Rad) with the
qPCR core kit for SYBR green I (Eurogentec). PCR was performed in 96-
well optical reaction plates heated for 10 min to 958C to activate hot start
Taq DNA polymerase, followed by 50 cycles of denaturation for 60 s at
958C and annealing extension for 60 s at 588C. Target quantifications
were performed with specific primer pairs designed with Beacon De-
signer 4.0 (Premier Biosoft International). PCR experiments were per-
formed in triplicate. Expression levels were first normalized to ACTIN2
expression levels that did not show clear systematic changes in Ct value
and then to the respective expression levels in the NPA-grown roots.
The primers used to quantify gene expression levels were as follows:
At3g18780/ACTIN2, 59-TTGACTACGAGCAGGAGATGG-39 and 59-ACA-
AACGAGGGCTGGAACAAG-39;At1g73590/PIN1,59-TACTCCGAGACC-
TTCCAACTACG-39 and 59-TCCACCGCCACCACTTCC-39; At5g57090/
PIN2, 59-GGCGAAGAAAGCAGGAAGA-39 and 59-GGTGGGTACGACG-
GAACA-39; At1g70940/PIN3, 59-GAGGGAGAAGGAAGAAAGGGAAAC-39
and 59-CTTGGCTTGTAATGTTGGCATCAG-39; At2g01420/PIN4, 59-ATG-
CTGGTCTTGGAATGGCTATG-39 and 59- CTGAACGATGGCTATACGG-
AGAAG-39; At1g77110/PIN6, 59-CCACGGCGGAGGAGGAAG-39 and
59-AGTAAGCATCGGAGGAAGCATAAC-39; At1g23080/PIN7, 59-ACT-
CCTCGTCCGTCTAATCTCAC-39 and 59-GAAGCCATAGCACAACTCTC-
CTC-39.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. BAP Inhibits Root Growth and Lateral Root
Formation.
Supplemental Figure 2. The Inhibitor of Ethylene Biosynthesis AVG
Prevents Cytokinin Inhibition of Primary Root Growth but Does Not
Change the Effect of Cytokinins on Lateral Root Density.
Supplemental Figure 3. L1-Specific IPT Expression Recovers Cyto-
kinin Phenotypes.
Supplemental Figure 4. IPT Transactivation in Xylem-Pole Pericycle
Cells Does Not Change Cell Specification.
Supplemental Figure 5. IPT Transactivation in Young Lateral Root
Primordia Does Not Change Root Growth or Branching.
Supplemental Figure 6. IPT Transactivation in Xylem-Pole Pericycle
Cells Leads to the Formation of Disorganized Primordia.
Supplemental Figure 7. Cytokinin Treatment Does Not Change
Auxin Sensitivity in Root Xylem-Pole Pericycle Cells.
Supplemental Figure 8. PIN Gene Expression Is Strongly Reduced in
the Shoot of Cytokinin-Treated Plants.
ACKNOWLEDGMENTS
This work was funded by the Institut de Recherche pour le De ´veloppe-
ment (L.L. and D.B.), the Biotechnology and Biological Science Research
Council (R.S., N.G., and M.B.), Biotechnology and Biological Science
Research Council–Engineering and Physical Sciences Research Council
Centres for Integrative Systems Biology funding to the Centre for Plant
Integrative Biology (M.B.), the Margarete von Wrangell-Habilitationspro-
gramm (E.B.), the Consejeria de Educacion, Ciencia, y Technologia, Junta
de Extremadura (BRV010130 to I.C.), the Institute for the Promotion of
Innovation through Science and Technology in Flanders (S.V.), the Nether-
lands Organization for Scientific Research (D.W.), the Volkswagenstiftung
andEuropeanMolecularBiologyOrganization(J.F.),theInstitutNationalde
laRechercheAgronomique(P.D.),andaBritishCouncil/EgideAlliancegrant
(05752SMtoL.L.andM.B.).WethankJ.Coates(UniversityofBirmingham,
UK) for critical reading of the manuscript.
Received September 26, 2007; revised October 26, 2007; accepted
November 18, 2007; published December 7, 2007.
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