domain ii mutations in crane/iaa18 suppress lateral root
TRANSCRIPT
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
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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
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(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.
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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.
Regulation of lateral root formation by IAA18 1029
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(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.
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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.
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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.
1032 Regulation of lateral root formation by IAA18
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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.
Regulation of lateral root formation by IAA18 1033
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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).
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(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
Regulation of lateral root formation by IAA18 1035
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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
1036 Regulation of lateral root formation by IAA18
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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.
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(Received March 26, 2008; Accepted May 21, 2008)
1038 Regulation of lateral root formation by IAA18
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