Domain II Mutations in CRANE/IAA18 Suppress Lateral Root

Domain II Mutations in CRANE/IAA18 Suppress Lateral Root Formation

and Affect Shoot Development in Arabidopsis thaliana

Takeo Uehara1, Yoko Okushima

2, Tetsuro Mimura

3, Masao Tasaka

2and Hidehiro Fukaki

3,*

1 Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Kobe, 657-8501 Japan2 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0192 Japan3 Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Kobe, 657-8501 Japan

Lateral root formation is an important developmental

component of root systems in vascular plants. Several regu-

latory genes for lateral root formation have been identified

from recent studies mainly using Arabidopsis thaliana. In this

study, we isolated two dominant mutant alleles, crane-1 and

crane-2, which are defective in lateral root formation in

Arabidopsis. The crane mutants have dramatically reduced

lateral root and auxin-induced lateral root formation, indicat-

ing that the crane mutations reduce auxin sensitivity. In

addition, the crane mutants have pleiotropic phenotypes in the

aerial shoots, including long hypocotyls when grown in the

light, up-curled leaves and reduced fertility. The crane mutant

phenotypes are caused by a gain-of-function mutation in

domain II of IAA18, a member of the Aux/IAA transcrip-

tional repressor family which is expressed in almost all organs.

In roots, IAA18 promoter::GUS was expressed in the early

stages of lateral root development. In the yeast two-hybrid

system, IAA18 interacts with AUXIN RESPONSE

FACTOR 7 (ARF7) and ARF19, transcriptional activators

that positively regulate lateral root formation. Taken

together, our results indicate that CRANE/IAA18 is involved

in lateral root formation in Arabidopsis, and suggest that it

negatively regulates the activity of ARF7 and ARF19 for

lateral root formation.

Keywords: Arabidopsis — Aux/IAA genes — Auxin —

IAA18 — Lateral root formation.

Abbreviations: AFB, AUXIN RECEPTOR F-BOXPROTEIN; ARF, AUXIN RESPONSE FACTOR; ASL,ASYMMETRIC LEAVES2-LIKE; Aux/IAA, AUXIN/INDOLE-3-ACETIC ACID; AuxRE, auxin response element;CTD, C-terminal domain; EMS, ethyl methanesulfonate; GUS,b-glucuronidase; LBD, LATERAL ORGAN BOUNDARIES-DOMAIN; NAA, naphthaleneacetic acid; NPA, N-1-naph-thylphthalamic acid; RT–PCR, reverse transcription–PCR; slr,solitary-root; TIR1, TRANSPORT INHIBITOR RESPONSE1.

Introduction

Lateral root formation is one of the most important

events in the development of the root system of vascular

plants (Osmont et al. 2007). Lateral roots are branched

secondary roots that are produced de novo from the

primary root or parental roots. Formation of lateral roots

under adequate regulatory control results in the construc-

tion of a root system that supports the plant body and

absorbs water and nutrients from soil. Formation of a

lateral root primordium is initiated by the highly regulated

division of cells in pericycle files of the primary root in most

plant species (Barlow et al. 2004). This initiation event is a

key step of lateral root formation, but the regulatory mech-

anisms that control lateral root (primordium) formation

are largely unknown.

The plant hormone auxin is known to be an important

regulatory agent for plant growth and development, includ-

ing lateral root formation, embryonic development, tropic

responses, apical dominance and vascular development

(Woodward and Bartel 2005). In several plant species,

exogenously applied auxin promotes pericycle cell division,

and causes lateral root formation (Torrey 1950, Blakely

et al. 1988, Laskowski et al. 1995), and inhibitors of auxin

transport, such as N-1-naphthylphthalamic acid (NPA),

suppress the initiation of lateral root primordia (Reed et al.

1998b, Casimiro et al. 2001). Moreover, many Arabidopsis

mutants which are impaired in auxin biosynthesis, homeo-

stasis, transport or signaling have fewer lateral roots than

the wild type (Fukaki et al. 2007). These observations

indicate that auxin is a key regulator of lateral root

formation. However, the molecular mechanisms of auxin

action in this process are not fully understood.

At the cellular level, auxin regulates the transcription

of many genes. Exogenous auxin application rapidly and

transiently induces the transcription of early genes such

as members of the AUXIN/INDOLE-3-ACETIC ACID

(Aux/IAA), SMALL AUXIN-UP RNA (SAUR) and

Gretchen Hagen3 (GH3) gene families (Abel and Theologis

1996). The Aux/IAA and AUXIN RESPONSE FACTOR

(ARF) transcription regulator families mediate expression

of auxin-regulated gene expression (Guilfoyle and Hagen

2007), and 29 Aux/IAA genes have been identified in the

Arabidopsis genome (Reed 2001). Expression of each Aux/

IAA gene is regulated in a specific temporal and spatial

manner, strongly suggesting that Aux/IAA genes have

*Corresponding author: E-mail, [email protected]; Fax, þ81-78-803-5721

Plant Cell Physiol. 49(7): 1025–1038 (2008)doi:10.1093/pcp/pcn079, available online at www.pcp.oxfordjournals.org� The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

1025

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distinct and overlapping functions in auxin signaling

(Teale et al. 2006).

Aux/IAA proteins have four highly conserved domains

(domains I–IV). Domains III and IV are similar to the

C-terminal domains (CTDs) of ARF proteins. ARFs can

bind to auxin response elements (AuxREs) in the promoter

regions of early auxin response genes and activate or repress

their transcription. Domains III and IV mediate homo- or

heterodimerization between two Aux/IAA proteins or bet-

ween Aux/IAA and ARF. Domain I is similar to the

conserved domain of the ethylene response factor (ERF)

transcriptional repressors, and can inactivate the transcrip-

tional activity of ARFs (Tiwari et al. 2004). Aux/IAAs thus

negatively modulate auxin-regulated gene expression as

transcriptional repressors via this interaction. Domain II of

Aux/IAA contributes to the instability of this protein.

Several gain-of-function mutants, which have missense

mutations in domain II, have been identified, such as

iaa1/axr5 (Yang et al. 2004), iaa3/shy2 (Tian and Reed

1999), iaa7/axr2 (Nagpal et al. 2000), iaa12/bdl (Hamann

et al. 2002), iaa14/slr (Fukaki et al. 2002), iaa17/axr3

(Rouse et al. 1998), iaa19/msg2 (Tatematsu et al. 2004) and

iaa28 (Rogg et al. 2001). These gain-of-function mutations

stabilize the Aux/IAA protein, thereby causing a variety of

auxin-related phenotypes (Woodward and Bartel 2005). In

the wild type, Aux/IAA proteins are degradated by the

ubiquitin–proteasome pathway (Gray et al. 2001). The

auxin receptors, TRANSPORT INHIBITOR RESPONSE1

(TIR1) and three TIR1-related AUXIN RECEPTOR F-

BOX PROTEINs (AFBs), are subunits of the SCFTIR1/AFBs

ubiquitin-ligase complex and act as F-box proteins for

recruiting Aux/IAA proteins in Arabidopsis (Dharmasiri

et al. 2005, Kepinski and Leyser 2005). The interaction

between Aux/IAAs and these F-box proteins is mediated by

the Aux/IAA domain II. The missense mutations in domain

II inhibit the interaction between Aux/IAA and TIR1/

AFBs, thus the mutated Aux/IAA proteins are not

degradated, resulting in constitutive inactivation of ARF

transcriptional activity.

We previously identified a gain-of-function mutant

solitary-root (slr), which has a missense mutation in domain

II of IAA14 (Fukaki et al. 2002). The slr mutant has no

lateral roots, few root hairs and abnormal gravitropic

responses in roots and hypocotyls, strongly suggesting

that stabilized mutant IAA14 protein inactivates the

transcriptional activity of the ARFs that are responsible

for these auxin-mediated developmental processes. The

observation that arf 7 arf19 double mutants are very similar

to the iaa14/slr mutant in lateral root formation and root

gravitropic response (Okushima et al. 2005, Wilmoth et al.

2005) indicates that IAA14, ARF7 and ARF19 probably

function in the same genetic pathway of Arabidopsis

development. In addition, iaa3/shy2, iaa19/msg2 and

iaa28-1 mutants also have reduced numbers of lateral

roots, although the phenotypes of these mutants are not as

severe as iaa14/slr in terms of lateral root formation. ARF7

and ARF19 interact with several Aux/IAAs including SLR/

IAA14, SHY2/IAA3, MSG2/IAA19 and IAA28 in yeasts

(Tatematsu et al. 2004, Fukaki et al. 2005, Y. Okushima

and H. Fukaki unpublished results). SLR/IAA14 and the

other Aux/IAAs thus are likely to be negative regulators

of ARF7 and ARF19 through this interaction, thereby

negatively regulating lateral root formation. However, it is

unknown how many Aux/IAA members regulate lateral

root formation because no gain-of-function mutants have

been characterized in more than half of the Aux/IAA

proteins.

Here, we report the isolation and characterization of

two novel Arabidopsis mutant alleles, crane-1 and crane-2,

which cause defective lateral root formation. These two

mutants are dominant and have almost identical phenotypic

characteristics, such as up-curled leaves, dwarfism and low

fertility. In crane mutants, lateral root formation is blocked

in very early developmental stages. Both crane mutants are

due to gain-of-function mutations in IAA18, a member of

Aux/IAA gene family. IAA18 is expressed in almost all

organs of Arabidopsis, and the promoter activity of IAA18

was strong in the early stages of lateral root formation.

In addition, we confirmed that IAA18 protein interacts

directly with ARF7 and ARF19 in yeast. Our results

indicate that CRANE/IAA18 is involved in lateral root

formation and suggest that it functions cooperatively with

SLR/IAA14 as a repressor of ARF7 and ARF19.

Results

Isolation of crane mutants

Although the arf 7 arf19 double mutant has an obvious

lateral root formation deficiency phenotype (Okushima

et al. 2005), each of the single mutants, arf 7 and arf19, has

milder or almost no abnormal phenotype in lateral root

formation, presumably because of the functional redun-

dancy between ARF7 and ARF19 (Tatematsu et al. 2004,

Okushima et al. 2005, Wilmoth et al. 2005). To identify

novel factors that cooperatively regulate lateral root

formation with ARF7 and ARF19, we screened ethyl

methanesulfonate (EMS)-mutagenized M2 seedlings for

enhancers of the arf 7-1 and arf19-4 single mutants.

About 150 M2 lines were affected in lateral root

formation, and 430 of them were double mutants with

a previously known mutation, such as shy2/iaa3, slr/iaa14,

tir3, arf 7 (for arf19-4) and arf19 (for arf 7-1), which have

been reported to affect lateral root formation (data not

shown). Among the remainder, we identified two indepen-

dent lines from arf 7-1 and arf19-4, respectively, which had

almost the same phenotypes as each other, but which did

1026 Regulation of lateral root formation by IAA18


not resemble mutants that had been described previously.

We named these two mutants crane-1 and crane-2,

respectively, after the origami crane bird. Genetic analysis

indicated that the crane-1 and crane-2 mutations are

dominant. Even after the backcrossing away from the

arf 7-1 or arf19-4 mutation, each crane mutation caused

almost identical phenotypes to the original crane-1 arf 7-1

and crane-2 arf19-4 double mutants. Therefore, we analyzed

the effects of the single crane mutations on lateral root

formation and other developmental events, rather than as

a genetic enhancer of arf 7 or arf19.

crane mutants show reduced lateral root formation

The primary roots of 15-day-old light-grown wild-type

Col seedlings produced many lateral roots, which are drama-

tically reduced in crane-1 and crane-2 (Fig. 1A). In 8-day-old

seedlings, the length of primary roots was 51.0� 8.5mm

(mean� SD) for Col and 39.5� 9.5mm for crane-2

A B

C D

F G H

E

Colcrane-1

crane-2

slr-1

Col crane-2

Col

Col

crane-2

crane-2

Col crane-2

6

hypo

coty

l len

gth

(mm

)

5

4

3

2

1

0Col crane-2

Fig. 1 Phenotypes of wild-type Columbia (Col) and crane mutants. (A) Fifteen-day-old seedlings of Col, crane-1, crane-2 and slr-1/iaa14.Lateral root formation of crane mutants is drastically reduced. (B) One-month-old Col and crane-2 plants. The crane mutants are dwarfed.(C, D) The aerial parts of 15-day-old seedlings of Col (C) and crane-2 (D). cranemutant hypocotyls are longer than wild-type Col. (E) Rosetteleaves of Col and crane-2. In crane mutants, the rosette leaves are up-curled. (F, G) Flowers of Col (F) and crane-2 (G) plants. Stamens ofcrane-2 are shorter than those of Col. Scale bars represent 10mm for A, B and E, and 20mm for C and D. (H) Hypocotyl lengths of 8-day-oldCol (n¼ 10) and crane-2 (n¼ 10) seedlings. Hypocotyls of crane-2 are about twice as long as those of Col. Error bars represent the SD.

Regulation of lateral root formation by IAA18 1027


(Fig. 2A), and the total number of lateral roots was 9.0� 2.8

for Col and 2.2� 1.8 for crane-2 (Fig. 2B). Lateral root

density, which is the number of lateral roots cm�1 primary

root, was 1.8� 0.6 for Col and 0.6� 0.5 for crane-2 (Fig. 2C).

The aerial parts of these crane mutants also have

several distinctive phenotypes (Fig. 1B–H). The cotyledons

and rosette leaves of Col are almost flat or slightly down-

curled, whereas they are up-curled on the crane mutants

(Fig. 1C–E). In addition, crane mutants are dwarfed

(Fig. 1B), have longer hypocotyls (Fig. 1C, D, H), shorter

stamens (Fig. 1F, G) and lower fertility rates (data not

shown) than the wild type. The gravitropic responses of

seedling roots and hypocotyls are not dramatically affected

by the crane-2 mutation (Supplementary Fig. S1).

The crane mutation blocks the early stages in lateral root

formation

In Arabidopsis, the development of a lateral root is

described in nine stages (Malamy and Benfey 1997). The

formation of a lateral root primordium is initiated by

pericycle cell division in a transverse orientation (anticlinal

cell division, stage I). Subsequently, some of the divided

cells in stage I divide in a longitudinal orientation

(periclinal cell division) that produce two layers from a

single pericycle cell layer (stage II). After stage II, the lateral

root primordium is formed through further cell division

and differentiation (stages III and IV). To determine which

stages of lateral root development are affected in the crane

mutants, we analyzed the expression of a cell cycle marker

gene, pCycB1;1::CycB1;1(NT)-GUS, in the crane-2 pri-

mary root. In the pCycB1;1::CycB1;1(NT)-GUS line, the

chimeric protein containing the destruction box of CycB1;1

fused to b-glucuronidase (GUS) is expressed under the

control of the CycB1;1 promoter (Colon-Carmona et al.

1999). As CycB1;1 is expressed only at around the G2–M

transition of the cell cycle (Doerner et al. 1996, Fukaki et al.

2002), GUS activity (foci) of 8-day-old light-grown

pCycB1;1::CycB1;1(NT)-GUS seedlings was detected in

the dividing cells of the root pericycle where lateral root

initiation occurs, as well as in the primary root apical

meristem (Fig. 3A, B, E). In contrast, the 8-day-old light-

grown pCycB1;1::CycB1;1(NT)-GUS/crane-2 seedlings

have a smaller number of GUS foci around the root

pericycle cells than pCycB1;1::CycB1;1(NT)-GUS/Col

seedlings, although foci were detected at the primary root

apical meristem (Fig. 3C, E). These observations indicate

that the crane mutation reduces the division of pericycle

cells in the early stages of lateral root initiation, probably

the first anticlinal cell division of the pericycle.

Auxin sensitivity of crane mutant roots

The crane mutant in lateral root formation and other

developmental phenotypes indicates that the cranemutation

Fig. 2 Root phenotypes of Col (n¼ 23) and crane-2 (n¼ 29)seedlings. (A) Primary root lengths (mm). (B) Number of lateral rootsformed per plant. (C) Lateral root density is the number of lateralroots per linear cm primary root (cm�1). Differences in lateral rootdensity between 8-day-old seedlings of Col and crane-2 arestatistically significant (P50.01, Student’s t-test). Error barsrepresent the SD.



alters sensitivity to auxin. To investigate this possibility, we

examined the effects of exogenous auxin, naphthaleneacetic

acid (NAA), on crane-2 root growth. Five-day-old Col and

crane seedlings grown on NAA-free medium were trans-

ferred to NAA-free or NAA-containing medium and

incubated for an additional 4 d. In both Col and crane-2,

0.1 mM NAA reduced the growth of primary roots to a

similar extent [32.0� 7.1% of control in Col; 43.0� 19.1%

of control in crane-2 (n¼ 12); no significant difference was

observed between Col and crane-2. (P40.05, by Student’s

t-test)] (Fig. 4A). In contrast, Col seedlings treated with

0.1 mM NAA formed 410 lateral roots per individual

D

E

A B C

4

3

2

Num

ber

of in

divi

dual

s

1

0

3

2

Num

ber

of in

divi

dual

s

1

0

0 1 2 3 4 5

Number of emerged LR

6 7 8 9 10 11 12

0 1 2 3 4 5

Number of GUS foci

6 7 8 9 10 11 1312

Colcrane-2

Colcrane-2

Fig. 3 Expression of pCycB1;1::CycB1;1(NT)-GUS and lateral root formation in 8-day-old seedlings of Col and crane-2. (A, B)pCycB1;1::CycB1;1(NT)-GUS expression in Col primary roots. GUS-stained foci are observed at the root apical meristem (A) and in theearly stages of lateral root primordium formation (B). (C) pCycB1;1::CycB1;1(NT)-GUS expression in crane-2 primary roots. The GUS signalis observed in the primary root meristem. Scale bars represent 100 mm. (D, E) Emerged lateral root (LR) counts (D) and lateral root primordiavisualized as GUS foci (E) in Col (n¼ 14) and crane-2 (n¼ 13). The y-axis represents the numbers of individuals; the x-axis represents thenumbers of emerged LRs or GUS foci.



(n¼ 12) whereas the crane-2 seedlings treated with 0.1 mMNAA formed 6.3 lateral roots per individual (n¼ 12)

(P50.01, by Student’s t-test), indicating that the crane-2

mutant has reduced sensitivity to 0.1 mM NAA in auxin-

induced lateral root formation (Fig. 4B). However, 1 mMNAA inhibited primary root growth and induced lateral

root formation in the crane-2 mutant similar to Col

(Fig. 4A, B). In contrast, as described previously (Fukaki

et al. 2002), slr-1/iaa14 was more resistant to exogenous

0.1 mM NAA in the root growth inhibition assay (Fig. 4A)

and produced almost no lateral roots in response to 1 mMNAA (Fig. 4B). These observations indicate that the auxin

sensitivity of crane-2 roots is attenuated but not as inhibited

as in the slr-1 mutant.

DR5::GUS is a useful reporter gene for studies on

auxin-responsive gene transcription in Arabidopsis

(Ulmasov et al. 1997) and is widely used for visualizing

auxin response maxima; it can be useful for following

endogenous free auxin distribution (Benkova et al. 2003).

DR5::GUS expression was observed in the crane-2 back-

ground around the distal regions of primary and lateral

roots (Fig. 4C–F) and was enhanced by treatment

with 1mM NAA (data not shown) as observed in the

wild-type Col.

CRANE encodes IAA18 protein

As described above, some of the pleiotropic pheno-

types characteristic of crane mutants are observed among

the dominant mutants of Aux/IAA genes such as slr/iaa14

(Liscum and Reed 2002). Among the 29 Aux/IAA genes

found in the Arabidopsis genome, the mutants axr5/iaa1

(Yang et al. 2004), shy2/iaa3 (Tian and Reed 1999), bdl/

iaa12 (Hamann et al. 2002), slr/iaa14 (Fukaki et al. 2002),

axr3/iaa17 (Rouse et al. 1998), msg2/iaa19 (Tatematsu et al.

2004) and iaa28 (Rogg et al. 2001) have been reported

previously. All these mutants have an amino acid alteration

A

C D E F

B

100

80

prim

ary

root

gro

wth

(%

)

60

40

20

0

25

20

emer

ged

late

ral r

oots

15

10

5

00.1µM NAA

Col crane-2

1µM NAA control 0.1µM

NAA

1µM

***

*

***

Colcrane-2slr-1

Colcrane-2slr-1

Fig. 4 Auxin sensitivity of wild-type Col, crane-2 and slr-1/iaa14 roots. (A, B) Col, crane-2 and slr-1 seedlings were grown on NAA-freeMS solid medium for 5 d, transferred to NAA-free or NAA-containing medium, and grown for an additional 4 d. (A) Root elongation ofseedlings on 0.1 and 1 mM NAA medium for 4 d. Data are represented as growth of primary roots relative to growth on NAA-free medium.Error bars represent the SD. �P50.05, ��P50.001 (Student’s t-test). (B) Numbers of emerged lateral roots after 4 d of treatment. Error barsrepresent the SD. �P50.01, ��P50.001 compared with Col (Student’s t-test). (C–F) DR5::GUS expression in wild-type Col (C and D) andcrane-2 (E and F). DR5::GUS expression in root apical meristem (E) and lateral root primordium (F) was not affected in crane-2. Scale barsrepresent 100mm for C and E, and 50 mm for D and F.



in domain II of their respective Aux/IAA protein, but no

mutants like crane with the phenotypes including long

hypocotyls, up-curled leaves and reduced lateral root

formation had been reported. The crane locus could not

be genetically mapped because not enough F2 seeds were

produced due to the very low fertility of F1 plants from a

cross between crane and Ler. Therefore, in order to isolate

the crane gene, the DNA sequences of IAA2, 4, 5, 8, 9, 10,

11, 13, 15, 16, 18, 26, 27, 29, 31, 32, 33 and 34 were

compared in wild-type Col, crane-1 and crane-2 genomic

DNA. Both crane-1 and crane-2 have a single-nucleotide

missense mutation in IAA18, a member of the Aux/IAA

genes (Fig. 5A).

The IAA18 protein consists of 267 amino acids and has

four conserved domains among all of the Aux/IAA proteins

(Fig. 5A). The crane-1 and crane-2 mutations cause the

Gly99Arg and Gly99Glu substitutions in domain II,

respectively (Fig. 5A).

To confirm that CRANE encodes IAA18, we examined

whether the 4 kbp genomic fragment containing IAA18 with

the crane-2 mutation gives the crane phenotypes in the wild-

type background (see Supplementary Fig. S2). Among four

independent transgenic lines harboring the 4 kbp fragment,

three lines had reduced lateral root formation compared

with the non-transgenic controls (Fig. 6A). In addition,

these lines had the same developmental abnormalities as

crane (Fig. 6A–D and data not shown).

Previously, a Gly99Glu substitution in domain II

(i.e. crane-2) was reported as iaa18-1 in unpublished results

by Nagpal and Reed (Reed 2001). The iaa18-1 allele was not

described in detail but defects in cotyledon phyllotaxy, long

hypocotyl, short root and up-curled leaves were associated

with the mutant (Reed 2001, Liscum and Reed 2002).

However, it has not been confirmed whether the iaa18-1

phenotypes are caused by the domain II mutation in the

IAA18 gene, but our observation that the two cranemutants

have long hypocotyls, short primary roots and up-curled

true leaves, as well as fused cotyledons at a very low rate

and that the crane phenotypes are caused by the domain II

mutation in IAA18 indicates that the iaa18-1 and crane

mutants are co-allelic and that they are gain-of-function

mutants in IAA18.

To examine the function of the IAA18 gene in the wild

type, we analyzed the GABI-Kat line 800H03, which has a

T-DNA insertion at the end of the third exon of the IAA18

coding region. No obvious phenotype was observed in this

line (data not shown), suggesting that IAA18 functions

redundantly with other Aux/IAAs, as is the case with Aux/

IAA genes (Overvoorde et al. 2005).

Expression of the IAA18 gene

Quantitative reverse transcription–PCR (RT–PCR)

was used to determine the expression pattern of IAA18

in five different organs of Arabidopsis. IAA18 was expressed

in the aerial parts and roots of seedlings, rosette leaves,

cauline leaves and inflorescences, though expression in roots

was lower than in other organs (Fig. 7). This result

is consistent with other published microarray data

(Zimmermann et al. 2004, Schmid et al. 2005).

We also examined the IAA18 expression pattern

using transgenic plants harboring GUS fused to the 2.0 kb

upstream region of IAA18 (pIAA18::GUS, see Supple-

mentary Fig. S2). In the pIAA18::GUS seedlings,

Fig. 5 The IAA18 protein and mutations. (A) Structure of IAA18protein with its four conserved domains and the single nucleotidesubstitution mutation in domain II that causes Gly99Arg in crane-1and Gly99Glu in crane-2. The black triangle represents the insertionsite of T-DNA in the GABI 800H30 line. (B) Amino acid sequencesof domain II of Aux/IAA proteins in the crane mutants and knownAux/IAA mutants (axr5-1, Yang et al. 2004; shy2-1, -2, -3, Tian andReed 1999; shy2-6, Fukaki et al. unpublished; axr2-1, Nagpal et al.2000; bodenlos, Hamann et al. 2002; slr-1, Fukaki et al. 2002;slr-2, -3, -4, Fukaki et al. unpublished; axr3-1, -3, Rouse et al. 1998;axr3-101, Okushima et al. unpublished; msg2-1, -2, -3, -4,Tatematsu et al. 2004; iaa28-1, Rogg et al. 2001). Domain II ishighly conserved among Aux/IAA proteins.



GUS expression was observed only in the pericycle

where lateral roots are initiated and only during the early

stages of lateral root development (Fig. 7B, D, E). No GUS

staining was observed in emerged lateral roots or in the

primary root meristem (Fig. 7C, F, G). GUS expression in

the pericycle was enhanced by 1mM auxin treatment for 5 h

(Fig. 7H), suggesting that IAA18 is expressed in an auxin-

dependent manner. In the pIAA18::GUS seedlings, neither

cotyledons nor true leaves had apparent GUS staining

(data not shown), suggesting that the upstream region used

in this experiment may not be sufficient to allow the

complete native expression pattern of IAA18, like the case

of the pIAA14::GUS line (Fukaki et al. 2002).

CRANE/IAA18 interacts with ARF7 and ARF19 in yeast

SLR/IAA14 functions cooperatively with ARF7 and

ARF19 for initiating lateral root formation, and IAA14

protein interacts directly with ARF7 and ARF19 in a yeast

A B

C D

4-kbT1 #1

4-kb T1 #1

4-kb T2 #2 4-kb T2 #4Col

Fig. 6 Phenotypes of transgenic plants with a 4 kbp genomic fragment containing the IAA18 gene with the crane-2 mutation (seeSupplementary Fig. S2). (A, B) T1 generation seedling and adult plants of line 1. (A) A 5-week-old seedling. (B) A 10-week-old plant. Line 1plants had severe phenotypes in both shoots and roots, and produced no T2 seeds. (C) T2 generation 6-day-old seedling of line 2. The line 2seedling had crane-like up-curled cotyledons. (D) Seven-day-old seedlings of Col and a T2 of line 4. Scale bars represent 10mm.



A

B

C

F

D

G

E

H

8

7

6

5

Rel

ativ

e ex

pres

sion

leve

l(I

AA

18/A

CT

2)

4

3

2

1

0

seedling

shoo

t

root

rose

tte le

af

caul

ine

leaf

flow

er

Fig. 7 Expression of the IAA18 gene. (A) Expression level of IAA18 in several tissues of 11-day-old seedlings and about 3-week-old wild-type plants. Relative expression levels are normalized to the expression of ACT2. Two biological duplicates and two independent samplesfor each biological duplicate were examined. Error bars represent sthe SD. (B) A 9-day-old pIAA18::GUS seedling. Lateral root primordia inthe early stages are specifically stained (arrows). (C–G) pIAA18::GUS signal is observed in the early stages of the lateral root primordium(D, E). The signal is diminished in the late stages of the lateral root primordium (F) and emerged lateral root (G). (H) Auxin-inducedexpression of pIAA18::GUS. A 7-day-old pIAA18::GUS seedling was transferred to a 1mM NAA MS plate, incubated for 5 h, and stained.Expression in the pericycle is enhanced by NAA treatment. Scale bars represent 50 mm for C–G and 100 mm for H.



two-hybrid system (Fukaki et al. 2005). IAA18 is co-expres-

sed with ARF7 and ARF19 at the lateral root initiation site

(Fig. 7; Okushima et al. 2005), suggesting that IAA18

interacts with ARF7 and ARF19. Yeast two-hybrid experi-

ments between IAA18 and ARF7 or ARF19 were per-

formed to determine if IAA18 protein also interacts with

ARF7 and ARF19. The results showed that IAA18 directly

interacts with both ARF7 and ARF19 in yeasts (Table 1),

suggesting that CRANE/IAA18 functions with ARF7 and

ARF19 for lateral root formation in planta.

Discussion

A gain-of-function mutation in CRANE/IAA18 blocks

lateral root formation

In this study, we identified gain-of-function mutants in

CRANE/IAA18, a gene which is involved in Arabidopsis

lateral root formation. Phenotypic analysis showed that the

crane mutation suppresses the initiation of lateral root

formation and attenuates auxin-induced lateral root for-

mation, suggesting that the crane mutation reduces auxin

sensitivity in the roots, thereby inhibiting lateral root

formation. The crane mutants also have pleiotropic

phenotypes in the aerial shoots, including long hypocotyl,

up-curled leaves and reduced fertility, indicating the

involvement of CRANE in shoot development. Molecular

genetic analysis revealed that the crane mutant phenotypes

are caused by a missense mutation in domain II of IAA18,

a member of the large Aux/IAA protein family, indicating

that CRANE encodes IAA18. Previously, Reed (2001)

reported a mutant (iaa18-1) that had the same amino acid

substitution in domain II of IAA18 as in crane-2. The iaa18-

1 mutant seedlings were briefly described as having long

hypocotyls and up-curled leaves (Reed 2001). Although no

lateral root phenotype was mentioned, the iaa18-1 mutant

phenotypes approximate those of the crane mutants, which

lends further support to the supposition that CRANE

encodes IAA18.

All of the previously reported gain-of-function muta-

tions of Aux/IAA genes are due to amino acid substitutions

within the five amino acids of domain II (GWPPV)

(see Fig. 5B). For example, the shy2-1/-2, axr2-1, bodenlos,

msg2-4 and iaa28-1 alleles have a mutation in the first

proline residue in domain II of IAA3, IAA7, IAA12, IAA19

and IAA28, respectively. Similarly, the shy2-6, axr3-1, slr-1

and msg2-1/3 alleles have mutations in the second proline of

IAA3, IAA17, IAA14 and IAA19, respectively. Apparently,

substitutions of either of the two proline residues results in

severe phenotypes. The glycine to glutamate mutation in

IAA3, shy2-3, results in a weak mutant phenotype

compared with shy2-2, the proline to serine mutation in

IAA3 (Reed et al. 1998a, Tian and Reed 1999). Similarly,

the glycine to glutamate mutation in IAA17 domain II,

axr3-101, which we found recently, caused milder mutant

phenotypes than the axr3-1 and axr3-3mutants, which have

mutations in one of the two proline residues of IAA17

domain II (Supplementary Fig. S3; Leyser et al. 1996,

Rouse et al. 1998). The fact that crane-1 and crane-2 have

glycine to arginine and glycine to glutamate substitutions in

the IAA18 domain II, respectively, suggests that a mutation

in either of the two proline residues of the IAA18 domain II

would result in a more severe phenotype than crane-1

and crane-2, although such crane alleles have not yet

been found.

Recently, the structural basis for the mechanism of

interaction of the Aux/IAA protein with TIR1, a compo-

nent of the auxin receptor, has been reported (Tan et al.

2007). In domain II, tryptophan and the second proline

interact with a hydrophobic wall of the TIR1 pocket and

pack the auxin molecule into the pocket. The first proline

maintains the relative position of these two residues.

Glycine is critical for flexibility between the N-terminal

region and domain II of the Aux/IAA protein. Both crane

mutations may thus affect the flexibility of the IAA18

protein and its degradation by the TIR1-mediated proteo-

lytic pathway.

Aux/IAA proteins have distinct and overlapping functions

in plant growth and development

Gain-of-function mutants of SLR/IAA14 (Fukaki et al.

2002), IAA28 (Rogg et al. 2001) and MSG2/IAA19

(Tatematsu et al. 2004) have lateral root formation

phenotypes that are similar to crane/iaa18. However, only

slr/iaa14 has no lateral roots (Fukaki et al. 2002), but iaa28,

msg2/iaa19 and crane/iaa18 initiate a few lateral roots.

There are several other phenotypic differences between

these mutants. For example, slr/iaa14 has increased apical

dominance, but iaa28 has decreased apical dominance

Table 1 Interaction between IAA18 and ARFs in the yeast

two-hybrid system

GAL4-AD GAL4-DBD b-Galactosidase

activity (Miller unit)

IAA18 ARF7-CTD 13.1� 1.58

IAA18 ARF19-CTD 13.5� 1.57

IAA18 Empty ND

Empty ARF7-CTD NDa

Empty ARF19-CTD NDa

Yeast cells transformed with the indicated plasmids were analyzedfor b-galactosidase activity. IAA18, full-length IAA18 fused to theGAL4 activation domain (AD); ARF7-CTD and ARF19-CTD,C-terminal regions (domains III and IV) of ARF7 and ARF19fused to the GAL4 DNA-binding domain (BD); empty, negativecontrol; ND, not detected. Values are the means� SD.a These data were reported previously by our group (Fukakiet al. 2005).



(Rogg et al. 2001, Fukaki et al. 2002). In addition, msg2/

iaa19 has a unique differential growth of hypocotyls

phenotype (Tatematsu et al. 2004) and, as is evident from

this work, crane/iaa18 also has unique hypocotyl elongation

and leaf growth phenotypes. These phenotypic differences

among the four mutants indicate that each Aux/IAA

protein has a distinct function in a variety of developmental

processes. One of the reasons for this variation is that the

expression patterns of most Aux/IAA genes, including

IAA14, IAA18, IAA19 and IAA28, are differentially regu-

lated in a tissue- and stage-specific manner (Rogg et al.

2001, Fukaki et al. 2002, Tatematsu et al. 2004, this study).

More interestingly, expression of different mutant Aux/IAA

proteins, including msg2-1/iaa19, axr2-1/iaa7 and slr-1/

iaa14 under the control of the same promoter, resulted

in different effects on Arabidopsis development, strongly

suggesting that these Aux/IAA proteins have functional

differences in repressing ARF activity (Muto et al. 2007).

Minor differences in the specificities of their expression

patterns and molecular functions would thus result in the

phenotypic differences observed among gain-of-function

mutants of Aux/IAA genes.

Interactions of Aux/IAAs with ARFs have been

investigated using the yeast two-hybrid system (Kim et al.

1997, Ulmasov et al. 1997, Ouellet et al. 2001, Hamann

et al. 2002, Hardtke et al. 2004, Tatematsu et al. 2004,

Fukaki et al. 2005, Weijers et al. 2005, Weijers et al. 2006),

in vitro pull-down assay (Tatematsu et al. 2004, Weijers

et al. 2006), co-immunoprecipitation in planta (Weijers

et al. 2006) and fluorescence cross-correlation spectroscopy

(Muto et al. 2006), but no differential interactions between

Aux/IAA proteins and ARFs have been reported in these

assays. On the other hand, Aux/IAA genes have the

specificities of their expression patterns (Abel and

Theologis 1996, Rogg et al. 2001, Fukaki et al. 2002,

Hamann et al. 2002, Tatematsu et al. 2004, Weijers et al.

2006, this study), and Aux/IAA proteins have functional

differences in repressing ARF activity (Weijers et al. 2006,

Muto et al. 2007). These data suggest that Aux/IAA

specificity depends on their expression pattern and their

functional differences in repressing ARF activity. However,

the possibility remains that any binding preference for

particular ARFs in planta affects Aux/IAA specificity.

IAA18 expression and function in root and shoot development

Expression analysis by quantitative RT–PCR showed

that IAA18 is highly expressed in the aerial organs of

Arabidopsis (Fig. 7). This is consistent with the pleiotropic

phenotypes observed in the crane mutants (see Fig. 1).

Up-curled true leaves were reported in the mutant allele of

NPH4/ARF7 (nph4-103) (Nakamoto et al. 2006). Thus, in

true leaves, IAA18 might repress NPH4/ARF7 activity.

A very similar phenotype to crane was also observed in true

leaves of plants with shy2/iaa3 alleles (Tian and Reed 1999)

and in a transgenic plant that expresses stabilized mutant

IAA26 protein (Padmanabhan et al. 2006). In addition, the

arf6 arf8 double mutant has shorter stamens than the wild

type (Nagpal et al. 2005), which are also observed in crane

mutants (Fig. 1G). This suggests that IAA18 represses

ARF6 and ARF8 activity in floral organs. Furthermore,

like the crane mutants, the arf8 single mutant grows longer

hypocotyls in the light (Tian et al. 2004) (Fig. 1C, D, H),

also suggesting that IAA18 represses ARF8 activity in

hypocotyls, thereby promoting hypocotyl elongation.

Our analysis using pIAA18::GUS transgenic plants

showed that IAA18 is expressed in the early stages of lateral

root primordium development (Fig. 7). In Arabidopsis,

SLR/IAA14, ARF7 and ARF19 are key transcriptional

regulators of lateral root formation (Fukaki et al. 2002,

Okushima et al. 2005, Wilmoth et al. 2005). SLR/IAA14 is

mainly expressed in the stele throughout the root elongation

and mature zones (Fukaki et al. 2002). ARF19 is also

expressed throughout root tissues, including the meriste-

matic region (Okushima et al. 2005). On the other hand,

expression of ARF7 is restricted to the stele and the early

stages of lateral root development (Okushima et al. 2005).

Thus, expression of IAA18 overlaps with that of ARF7 and

ARF19, in the stele, including in the pericycle where lateral

roots are initiated and in the early stages of lateral root

primordium. This suggests that IAA18, ARF7 and ARF19

function together to initiate lateral roots. The result from

the yeast two-hybrid experiment showing the interaction

between CRANE/IAA18 and ARF7 or ARF19 supports

this possibility (Table 1).

IAA18 is a negative regulator of lateral root formation

in Arabidopsis

In this study, we found that a transcriptional regulator,

CRANE/IAA18, is involved in lateral root formation in

Arabidopsis. A current model of auxin-mediated lateral root

formation in Arabidopsis would first show that in the

pericycle cells, where the lateral root is initiated, auxin is

perceived by the auxin receptor complex, SCFTIR1/AFBs.

Secondly, the ubiquitin–proteasome pathway mediated by

the SCFTIR1/AFBs complex and 26S proteasome degrades

Aux/IAA proteins including SLR/IAA14, CRANE/IAA18

and the other Aux/IAAs, thereby releasing active ARF7

and ARF19. Then, released ARF transcriptional activators

ARF7 and ARF19 induce the expression of genes required

for lateral root initiation such as LBD16/ASL18 and

LBD29/ASL16 (Okushima et al. 2007).

It is unclear which cell(s) uses CRANE/IAA18-

dependent auxin signaling during lateral root formation,

and which ARFs are negatively regulated by CRANE/

IAA18 in the other auxin-mediated developmental pro-

cesses, including hypocotyl growth, and shoot organ



development in Arabidopsis plants. To answer these

questions, further studies using techniques such as real-

time detection of the interactions between Aux/IAAs and

ARFs in planta will be necessary.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana wild-type Columbia (Col) andLandsberg erecta (Ler) accession, slr (Fukaki et al. 2002), arf 7-1(SALK_040394; Okushima et al. 2005) and arf19-4(SALK_009879, obtained from the Arabidopsis BiologicalResource Center; Wilmoth et al. 2005) lines were used.pCycB1;1::CycB1;1(NT)-GUS seeds were kindly supplied byPeter Doerner (University of Edinburgh, UK). DR5-GUS seedswere kindly supplied by Tom Guilfoyle (University of Missouri-Columbia, USA). The axr3-1 and axr3-3 mutant seeds were kindlysupplied by Ottoline Leyser (University of York, UK). Seeds weresurface-sterilized with 0.01% (v/v) Triton X-100 and 10% (v/v)sodium hypochlorite, and plated on Murashige–Skoog mediumcontaining 1.0% (w/v) sucrose solidified with 0.5% (w/v) gelangum or 1% (w/v) agar. Kanamycin and hygromycin were added toa final concentration of 50mg ml�1 from an aqueous 50mg ml�1

stock. Plates and plants transferred to soil were grown at 238Cunder constant white light.

Isolation of the crane-1 and crane-2 mutants

About 5,000 each of arf 7-1 and arf19-4 mutant seeds weremutagenized with EMS as described previously (Fukaki et al.2006). M2 seeds were harvested and sown on the MS plates toscreen for mutants with decreased numbers of lateral roots. Thecrane-1 and crane-2 mutants were isolated from arf 7-1 and arf19-4M2 populations, respectively. These crane arf double mutants werebackcrossed to the wild-type Col, and the resulting F1 generationwas crossed with Col to separate the crane mutation from the arfmutation. The arf 7-1 and arf19-4 mutations caused by T-DNAinsertion were detected by PCR with the LB-specific primer(50-GTTGCCCGTCTCACTGGTGAAAAG-30) and gene-specificprimers as follows: 50-TGTTGCACTCCTCTTTGAACC-30 and50-TGGACTTTTGCTGACCCACGA-30 for arf 7-1, and 50-GATGAACAGGAGAGAAGAAGC-30 and 50-GCGGTTGCATCGAGAAATCCT-30 for arf19-4.

CycB1;1::GUS and DR5::GUS expression in the crane-2 mutant

pCycB1;1::CycB1;1(NT)-GUS (Colon-Carmona et al. 1999)and DR5::GUS (Ulmasov et al. 1997) were crossed with crane-2 byartificial pollination. The resultant F2 seedlings expressing reportergenes were stained and observed as described previously (Malamyand Benfey 1997). For analysis of auxin-induced DR5::GUSexpression in crane-2, 7-day-old seedlings of DR5::GUS/crane-2were transferred to MS plates with or without 1 mM NAA,incubated for 4 h, and stained.

Auxin sensitivity of crane mutants

Five-day-old Col and crane seedlings grown on NAA-free MSplates were transferred to MS plates with or without 1 mM NAAand incubated for an additional 4 d. These plates were photo-graphed with a Nikon COOLPIX2500, and root length andnumber of lateral roots were tallied using ImageJ software(Abramoff et al. 2004) and NeuronJ plug-in (Meijering et al. 2004).

Cloning of the IAA18 gene

The genomic region containing domain II of the selected Aux/IAA genes (IAA2, 4, 5, 8, 9, 10, 11, 13, 15, 16, 18, 26, 27, 29, 31, 32,33, 34) was amplified from Col, crane-1 and crane-2 mutant DNAwith the primers listed in Supplementary Table S1. PCR productswere purified and directly sequenced by standard methods usingthese PCR primers and BigDye Terminator v3.0 (AppliedBiosystems, Lincoln Centre Drive Foster City, CA, USA).

IAA18 was amplified from Col cDNA using the primers:50-ATGGAGGGTTATTCAAGAAA-30 and 50-TCATCTTCTCATTTTCTCTT-30. The PCR product was cloned into the EcoRVsite of the pGreenII (Hellens et al. 2000) and pBluescriptSKþvectors (Fermentas, Burlington, Ontario, Canada), and confirmedby sequencing using the primers: M13(–20) (50-GTAAAACGACGGCCAGT-30) and M13RV (50-AGCGGATAACAATTTCACAC-30).

Transgenic plants expressing the mutated IAA18 gene

To produce the transgenic plants expressing the mutatedIAA18 gene, a 3,956 bp genomic region that spans IAA18 wasamplified from Col DNA using the primer set: 50-TATGATGGGATGGTAAATGG-30 and 50-CCCAAGAGTCCAGACAAAGA-30. The amplified fragment was cloned into the EcoRV siteof pBluescriptSKþ and subcloned into vector pBI301H, a deriva-tive of pBI101 (Clontech, Mountain View, CA, USA) made by theauthors, using the EcoRI and Sal I sites. This construct wasintroduced into Agrobacterium tumefaciens strain C58MP90 andtransformed into Col plants by the floral dipping method (Cloughand Bent 1998).

IAA18 expression

Total RNA was extracted from several Col seedling and adultplant organs using an RNeasy Plant Mini Kit (Qiagen, Valencia,CA, USA). cDNAs for each sample were synthesized by reversetranscription using Reverse Transcriptase XL (AMV) (Takara BioInc., Otsu, Shiga, Japan). IAA18 expression was analyzed usinga Smart Cycler

�II System (Cepheid, Sunnyvale, CA, USA) and

the primers: 50-TCGCTGGCAAATACTTCTCTC-30 and 50-CACTGGACCAGGAGCAGTTC-30. For an internal control,ACT2 expression was also analyzed using the primers: 50-TTGTTCCAGCCCTCGTTTGT-30 and 50-TCATGCTGCTTGGTGCAAGT-30.

A 2,003 bp fragment including the upstream region and 59 bpof open reading frame was amplified from Col genomic DNAusing the primer set: 50-TATGATGGGATGGTAAATGG-30 and50-GGAATCATCAAGTCAAGCAG-30. The amplified fragmentwas cloned into the SmaI site of pBI301. This construct wastransformed into Col plants as described above. T2 seedlings werestained and observed as described previously (Fukaki et al. 2002).

Interaction between IAA18 protein and ARF7 or ARF19 proteins

Yeast two-hybrid experiments between IAA18 and ARF7or ARF19 were performed as described previously (Fukakiet al. 2005). IAA18/pAD-GAL4-2.1 was constructed from aXhoI–PstI fragment of IAA18/pBluescriptSKþ and pAD-GAL4-2.1 (Stratagene, La Jolla, CA, USA).

Accession numbers and knockout line

Arabidopsis Genome Initiative locus identifiers for thegenes mentioned in this article are as follows: ARF7 (At5g20730),ARF19 (At1g19220), IAA14/SLR (At4g14550), IAA18/CRANE(At1g51950). GABI-Kat line 800H03 was obtained from



the Institute for Genome Research and Systems Biology (Rossoet al. 2003).

Supplementary material

Supplementary material mentioned in the article is avail-able to online subscribers at the journal website www.pcp.oxfordjournals.org.

Funding

The Scientific Research on Priority Areas (grants

17027019 and 19060006 to H.F.); Ministry of Education,

Culture, Sports, Science and Technology, Japan; the

Inamori Foundation (to H.F.); Hyogo Science and Tech-

nology Association (to H.F.); the Japan Society for the

Promotion of Science (research fellowship for young

scientists to Y.O.).

Acknowledgments

We thank Peter Doerner for pCycB1;1::CycB1;1(NT)-GUS,Tom Guilfoyle for DR5::GUS seeds, Ottoline Leyser for the axr3mutants, and the Arabidopsis Biological Resource Center for theseed stock. We gratefully thank Keiko Uno for her excellenttechnical assistance.

References

Abel, S. and Theologis, A. (1996) Early genes and auxin action. PlantPhysiol. 111: 9–17.

Abramoff, M.D., Magelhaes, P.J. and Ram, S.J. (2004) Image processingwith ImageJ. Biophot. Int. 11: 36–42.

Barlow, P.W., Volkmann, D. and Baluska, F. (2004) Polarity in roots. InPolarity in Plants. Edited by Lindsey, K. pp. 192–241. BlackwellPublishing, Oxford.

Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova, D.,Jurgens, G. and Frıml, J. (2003) Local, efflux-dependent auxin gradientsas a common module for plant organ formation. Cell 115: 591–602.

Blakely, L.M., Blakely, R.M., Colowit, P.M. and Elliott, D.S. (1988)Experimental studies on lateral root formation in radish seedling roots:II. Analysis of the dose–response to exogenous auxin. Plant Physiol. 87:414–419.

Casimiro, I., Marchant, A., Bhalerao, R.P., Beeckman, T., Dhooge, S.,Swarup, R., Graham, N., Inze, D., Sandberg, G., Casero, P.J. andBennett, M. (2001) Auxin transport promotes Arabidopsis lateral rootinitiation. Plant Cell 13: 843–852.

Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. Plant J.16: 735–743.

Colon-Carmona, A., You, R., Haimovitch-Gal, T. and Doerner, P. (1999)Technical advance: spatio-temporal analysis of mitotic activity with alabile cyclin–GUS fusion protein. Plant J. 20: 503–508.

Dharmasiri, N., Dharmasiri, S. and Estelle, M. (2005) The F-box proteinTIR1 is an auxin receptor. Nature 435: 441–445.

Doerner, P., Jørgensen, J.E., You, R., Steppuhn, J. and Lamb, C. (1996)Control of root growth and development by cyclin expression. Nature380: 520–523.

Fukaki, H., Nakao, Y., Okushima, Y., Theologis, A. and Tasaka, M. (2005)Tissue-specific expression of stabilized SOLITARY-ROOT/IAA14 alterslateral root development in Arabidopsis. Plant J. 44: 382–395.

Fukaki, H., Okushima, Y. and Tasaka, M. (2007) Auxin-mediated lateralroot formation in higher plants. Int. Rev. Cytol. 256: 111–137.

Fukaki, H., Tameda, S., Masuda, H. and Tasaka, M. (2002)Lateral root formation is blocked by a gain-of-function mutationin the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J. 29:153–168.

Fukaki, H., Taniguchi, N. and Tasaka, M. (2006) PICKLE is requiredfor SOLITARY-ROOT/IAA14-mediated repression of ARF7 andARF19 activity during Arabidopsis lateral root initiation. Plant J. 48:380–389.

Gray, W.M., Kepinski, S., Rouse, D., Leyser, O. and Estelle, M. (2001)Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins.Nature 414: 271–276.

Guilfoyle, T.J. and Hagen, G. (2007) Auxin response factors. Curr. Opin.Plant Biol. 10: 453–460.

Hamann, T., Benkova, E., Baurle, I., Kientz, M. and Jurgens, G. (2002) TheArabidopsis BODENLOS gene encodes an auxin response proteininhibiting MONOPTEROS-mediated embryo patterning. Genes Dev.

16: 1610–1615.Hardtke, C.S., Ckurshumova, W., Vidaurre, D.P., Singh, S.A.,

Stamatiou, G., Tiwari, S.B., Hagen, G., Guilfoyle, T.J. and Berleth, T.(2004) Overlapping and non-redundant functions of the Arabidopsisauxin response factors MONOPTEROS and NONPHOTOTROPIC

HYPOCOTYL 4. Development 131: 1089–1100.Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S. and

Mullineaux, P.M. (2000) pGreen: a versatile and flexible binaryTi vector for Agrobacterium-mediated plant transformation. Plant Mol.

Biol. 42: 819–832.Kepinski, S. and Leyser, O. (2005) The Arabidopsis F-box protein TIR1

is an auxin receptor. Nature 435: 446–451.Kim, J., Harter, K. and Theologis, A. (1997) Protein–protein interactions

among the Aux/IAA proteins. Proc. Natl Acad. Sci. USA 94:11786–11791.

Laskowski, M.J., Williams, M.E., Nusbaum, H.C. and Sussex, I.M. (1995)Formation of lateral root meristems is a two-stage process. Development

121: 3303–3310.Leyser, H.M., Pickett, F.B., Dharmasiri, S. and Estelle, M. (1996)

Mutations in the AXR3 gene of Arabidopsis result in altered auxinresponse including ectopic expression from the SAUR-AC1 promoter.Plant J. 10: 403–413.

Liscum, E. and Reed, J.W. (2002) Genetics of Aux/IAA and ARF actionin plant growth and development. Plant Mol. Biol. 49: 387–400.

Malamy, J.E. and Benfey, P.N. (1997) Organization and cell differentiationin lateral roots of Arabidopsis thaliana. Development 124: 33–44.

Meijering, E., Jacob, M., Sarria, J.C., Steiner, P., Hirling, H. and Unser, M.(2004) Design and validation of a tool for neurite tracing and analysis influorescence microscopy images. Cytometry A 58: 167–176.

Muto, H., Nagao, I., Demura, T., Fukuda, H., Kinjo, M. andYamamoto, K.T. (2006) Fluorescence cross-correlation analyses ofthe molecular interaction between an Aux/IAA protein, MSG2/IAA19and protein–protein interaction domains of auxin responsefactors of arabidopsis expressed in HeLa cells. Plant Cell Physiol. 47:1095–1101.

Muto, H., Watahiki, M.K., Nakamoto, D., Kinjo, M. andYamamoto, K.T. (2007) Specificity and similarity of functions of theAux/IAA genes in auxin signaling of Arabidopsis revealed by promoter-exchange experiments between MSG2/IAA19, AXR2/IAA3 and SLR/IAA14. Plant Physiol. 144: 187–196.

Nagpal, P., Ellis, C.M., Weber, H., Ploense, S.E., Barkawi, L.S., et al.(2005) Auxin response factors ARF6 and ARF8 promote jasmonic acidproduction and flower maturation. Development 132: 4107–4118.

Nagpal, P., Walker, L.M., Young, J.C., Sonawala, A., Timpte, C.,Estelle, M. and Reed, J.W. (2000) AXR2 encodes a member of theAux/IAA protein family. Plant Physiol. 123: 563–574.

Nakamoto, D., Ikeura, A., Asami, T. and Yamamoto, K.T. (2006)Inhibition of brassinosteroid biosynthesis by either a dwarf4 mutationor a brassinosteroid biosynthesis inhibitor rescues defects in tropicresponses of hypocotyls in the arabidopsis mutant nonphototropic

hypocotyl 4. Plant Physiol. 141: 456–464.Okushima, Y., Fukaki, H., Onoda, M., Theologis, A. and Tasaka, M.

(2007) ARF7 and ARF19 regulate lateral root formation via directactivation of LBD/ASL genes in Arabidopsis. Plant Cell 19: 118–130.



Okushima, Y., Overvoorde, P.J., Arima, K., Alonso, J.M., Chan, A., et al.(2005) Functional genomic analysis of the AUXIN RESPONSEFACTOR gene family members in Arabidopsis thaliana: unique andoverlapping functions of ARF7 and ARF19. Plant Cell 17: 444–463.

Osmont, K.S., Sibout, R. and Hardtke, C.S. (2007) Hiddenbranches: developments in root system architecture. Annu. Rev. PlantBiol. 58: 93–113.

Ouellet, F., Overvoorde, P.J. and Theologis, A. (2001) IAA17/AXR3: biochemical insight into an auxin mutant phenotype. Plant Cell13: 829–841.

Overvoorde, P.J., Okushima, Y., Alonso, J.M., Chan, A., Chang, C., et al.(2005) Functional genomic analysis of the AUXIN/INDOLE-3-ACETICACID gene family members in Arabidopsis thaliana. Plant Cell 17:3282–3300.

Padmanabhan, M.S., Shiferaw, H. and Culver, J.N. (2006) The Tobaccomosaic virus replicase protein disrupts the localization and function ofinteracting Aux/IAA proteins. Mol. Plant Microbe Interact. 19: 864–873.

Reed, J.W. (2001) Roles and activities of Aux/IAA proteins in Arabidopsis.Trends Plant Sci. 6: 420–425.

Reed, R.C., Brady, S.R. and Muday, G.K. (1998b) Inhibition of auxinmovement from the shoot into the root inhibits lateral root developmentin Arabidopsis. Plant Physiol. 118: 1369–1378.

Reed, J.W., Elumalai, R.P. and Chory, J. (1998a) Suppressors of anArabidopsis thaliana phyB mutation identify genes that control lightsignaling and hypocotyl elongation. Genetics 148: 1295–1310.

Rogg, L.E., Lasswell, J. and Bartel, B. (2001) A gain-of-function mutationin IAA28 suppresses lateral root development. Plant Cell 13: 465–480.

Rosso, M.G., Li, Y., Strizhov, N., Reiss, B., Dekker, K. and Weisshaar, B.(2003) An Arabidopsis thaliana T-DNA mutagenized population(GABI-Kat) for flanking sequence tag-based reverse genetics. PlantMol. Biol. 53: 247–259.

Rouse, D., Mackay, P., Stirnberg, P., Estelle, M. and Leyser, O. (1998)Changes in auxin response from mutations in an AUX/IAA gene. Science279: 1371–1373.

Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M.,Vingron, M., Scholkopf, B., Weigel, D. and Lohmann, J.U. (2005)A gene expression map of Arabidopsis thaliana development. Nat. Genet.37: 501–506.

Tan, X., Calderon-Villalobos, L.I., Sharon, M., Zheng, C., Robinson, C.V.,Estelle, M. and Zheng, N. (2007) Mechanism of auxin perception by theTIR1 ubiquitin ligase. Nature 446: 640–645.

Tatematsu, K., Kumagai, S., Muto, H., Sato, A., Watahiki, M.K.,Harper, R.M., Liscum, E. and Yamamoto, K.T. (2004) MASSUGU2

encodes Aux/IAA19, an auxin-regulated protein that functions togetherwith the transcriptional activator NPH4/ARF7 to regulate differentialgrowth responses of hypocotyl and formation of lateral roots inArabidopsis thaliana. Plant Cell 16: 379–393.

Teale, W.D., Paponov, I.A. and Palme, K. (2006) Auxin in action:signalling, transport and the control of plant growth and development.Nat. Rev. Mol. Cell Biol. 7: 847–859.

Tian, C.E., Muto, H., Higuchi, K., Matamura, T., Tatematsu, K.,Koshiba, T. and Yamamoto, K.T. (2004) Disruption and overexpressionof auxin response factor 8 gene of Arabidopsis affect hypocotyl elongationand root growth habit, indicating its possible involvement in auxinhomeostasis in light condition. Plant J. 40: 333–343.

Tian, Q. and Reed, J.W. (1999) Control of auxin-regulated rootdevelopment by the Arabidopsis thaliana SHY2/IAA3 gene.Development 126: 711–721.

Tiwari, S.B., Hagen, G. and Guilfoyle, T.J. (2004) Aux/IAA proteinscontain a potent transcriptional repression domain. Plant Cell 16:533–543.

Torrey, J.G. (1950) The induction of lateral roots by indoleacetic acid androot decapitation. Amer. J. Bot. 37: 257–263.

Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T.J. (1997) Aux/IAAproteins repress expression of reporter genes containing natural andhighly active synthetic auxin response elements. Plant Cell 9: 1963–1971.

Weijers, D., Benkova, E., Jager, K.E., Schlereth, A., Hamann, T.,Kientz, M., Wilmoth, J.C., Reed, J.W. and Jurgens, G. (2005)Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators. EMBO J. 24: 1874–1885.

Weijers, D., Schlereth, A., Ehrismann, J.S., Schwank, G., Kientz, M. andJurgens, G. (2006) Auxin triggers transient local signaling for cellspecification in Arabidopsis embryogenesis. Dev. Cell 10: 265–270.

Wilmoth, J.C., Wang, S., Tiwari, S.B., Joshi, A.D., Hagen, G.,Guilfoyle, T.J., Alonso, J.M., Ecker, J.R. and Reed, J.W. (2005)NPH4/ARF7 and ARF19 promote leaf expansion and auxin-inducedlateral root formation. Plant J. 43: 118–130.

Woodward, A.W. and Bartel, B. (2005) Auxin: regulation, action andinteraction. Ann. Bot. 95: 707–735.

Yang, X., Lee, S., So, J.H., Dharmasiri, S., Dharmasiri, N., Ge, L.,Jensen, C., Hangarter, R., Hobbie, L. and Estelle, M. (2004) The IAA1protein is encoded by AXR5 and is a substrate of SCFTIR1. Plant J. 40:772–782.

Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L. and Gruissem, W.(2004) GENEVESTIGATOR. Arabidopsis microarray database andanalysis toolbox. Plant Physiol. 136: 2621–2632.

(Received March 26, 2008; Accepted May 21, 2008)



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Domain II Mutations in CRANE/IAA18 Suppress Lateral Root