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Arabidopsis JAGGED LATERAL ORGANS Acts withASYMMETRIC LEAVES2 to
Coordinate KNOX and PINExpression in Shoot and Root Meristems C
W
Madlen I. Rasta,b and Rüdiger Simona,1
a Institut für Entwicklungsgenetik, Heinrich-Heine-Universität,
40225 Duesseldorf, GermanybDepartment of Plant Sciences, University
of Oxford, Oxford OX1 3RB, United Kingdom
Organ initiation requires the specification of a group of
founder cells at the flanks of the shoot apical meristem and
thecreation of a functional boundary that separates the incipient
primordia from the remainder of the meristem. Organdevelopment is
closely linked to the downregulation of class I KNOTTED1 LIKE
HOMEOBOX (KNOX) genes and accumulationof auxin at sites of
primordia initiation. Here, we show that Arabidopsis thaliana
JAGGED LATERAL ORGANS (JLO), a memberof the LATERAL ORGAN BOUNDARY
DOMAIN (LBD) gene family, is required for coordinated organ
development in shoot andfloral meristems. Loss of JLO function
results in ectopic expression of the KNOX genes SHOOT MERISTEMLESS
andBREVIPEDICELLUS (BP), indicating that JLO acts to restrict KNOX
expression. JLO acts in a trimeric protein complex withASYMMETRIC
LEAVES2 (AS2), another LBD protein, and AS1 to suppress BP
expression in lateral organs. In addition to itsrole in KNOX
regulation, we identified a role for AS2 in regulating PINFORMED
(PIN) expression and auxin transport fromembryogenesis onwards
together with JLO. We propose that different JLO and AS2 protein
complexes, possibly alsocomprising other LBD proteins, coordinate
auxin distribution and meristem function through the regulation of
KNOX and PINexpression during Arabidopsis development.
INTRODUCTION
Organ formation and growth requires a continuous supply ofnew
cells. The shoot apical meristem (SAM) of higher plants canprovide
these through a pool of pluripotent stem cells in thecentral zones.
When these stem cells divide, daughter cells aredisplaced toward
the periphery where they can be recruited toform organ primordia.
There, cells undergo rapid divisions, ex-pansion, and ultimately
differentiation. Cell fate appears to bedetermined mostly by a
cell’s position within the SAM. Emergingorgans are separated from
the remainder of the meristem bymorphological boundaries with
distinct cell division and geneexpression patterns (reviewed in
Rast and Simon, 2008).
Organ initiation in the peripheral zone is regulated by
twocritical events: the accumulation of auxin in a group of
foundercells and a simultaneous change in gene expression
programs.Such local auxin maxima are generated by active polar
transportmediated through auxin efflux carriers of the PINFORMED
(PIN)family (Galweiler et al., 1998; Paponov et al., 2005;
Zazimalovaet al., 2007). The direction of auxin flux within the L1
layer of theSAM is mainly determined by the subcellular
localization of PIN1(Benkova et al., 2003; Friml et al., 2003;
Reinhardt et al., 2003).
Live imaging of PIN1-GFP (for green fluorescent protein)
re-vealed the formation of an expression focus at the flanks of
theSAM that raises auxin levels in organ founder cells and
depletesauxin from the vicinity. As primordia growth starts, PIN1
polarityreverses to form a new auxin peak at a distant position.
Thus,phyllotactic pattering requires dynamic PIN1 polarity changes
togenerate new auxin peaks (Heisler et al., 2005).Meristematic and
organ founder cells are further distinguished
by the expression of specific gene sets. These contrasting
pat-terns depend on the mutual repression between meristem
andorgan-specific genes. For example, SHOOT MERISTEMLESS(STM), a
member of the class I KNOTTED LIKE HOMEOBOX(KNOX) family, is
specifically expressed in meristematic tissuesand excluded from
organ primordia. stmmutants lack a functionalSAM due to ectopic
expression of the MYB domain proteinASYMMETRIC LEAVES1 (AS1), which
is normally confined toorgan primordia (Byrne et al., 2000). AS1 in
turn restricts theKNOX genes BREVIPEDICELLUS (BP), KNAT2, and KNAT6
fromorgan initials (Belles-Boix et al., 2006; Byrne et al., 2000,
2002; Oriet al., 2000). This repression of KNOX genes depends on
themolecular interaction between AS1 and AS2, a protein that
be-longs to the LATERAL ORGAN BOUNDARY DOMAIN (LBD) familyof
Arabidopsis thaliana that shares the plant-specific LOB
domain(Shuai et al., 2002; Xu et al., 2003; Guo et al., 2008). All
LBDproteins analyzed localize to the nucleus (Iwakawa et al.,
2002;Borghi et al., 2007; Naito et al., 2007), and the LOB domain
wasshown to bind to DNA in vitro (Husbands et al., 2007).
Heteromersof AS1 and AS2 can directly interact with the promoter
regions ofBP and KNAT2. This binding is suggested to recruit the
chromatinremodeling factor HIRA, resulting in stable repression of
KNOXgenes in lateral organs (Phelps-Durr et al., 2005; Guo et al.,
2008).
1 Address correspondence to
[email protected] author responsible for
distribution of materials integral to the findingspresented in this
article in accordance with the policy described in theInstructions
for Authors (www.plantcell.org) is: Rüdiger Simon
([email protected]).C Some figures in this article
are displayed in color online but in black andwhite in the print
edition.WOnline version contains Web-only
data.www.plantcell.org/cgi/doi/10.1105/tpc.112.099978
The Plant Cell, Vol. 24: 2917–2933, July 2012, www.plantcell.org
ã 2012 American Society of Plant Biologists. All rights
reserved.
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The downregulation of KNOX genes in organ primordia
andauxin-regulated gene expression programs are
interconnected.Auxin activity appears to act in parallel with the
AS1/AS2 moduleto exclude BP expression from lateral organs (Hay et
al., 2006).Furthermore, leaf defects of plants that misexpress KNOX
genesare in part caused by the disruption of local auxin gradients
in theleaf margins (Tsiantis et al., 1999; Zgurski et al., 2005).
Correct cellfate allocation therefore requires the combined
activities of auxinand AS1/AS2 activity in the cells of lateral
organs and an antag-onistic activity of KNOX genes in meristematic
cells.
An important part of this regulation may take place at
theboundaries between organ primordia and the meristem (Aidaand
Tasaka, 2006). A number of boundary-specific genes wereshown to
contribute to meristem homeostasis and organ de-velopment. Among
them are CUP-SHAPED COTYLEDON1(CUC1), CUC2, and CUC3, which are
already required during theearly stages of embryogenesis to
activate and later delineateSTM expression (Aida et al., 1999).
Furthermore, boundary-specific expression of LATERAL ORGAN
BOUNDARIES (LOB),the founding member of the LBD gene family, is
promoted by BPand AS2 (Lin et al., 2003).
The LBD family gene JAGGED LATERAL ORGANS (JLO/LBD30) was
previously shown to be required for auxin-mediateddevelopment from
the earliest stages of embryogenesis on-wards. JLO loss-of-function
mutants (jlo-1 and jlo-2) arrestduring embryogenesis or early
seedling stages due to aberrantcell division patterns and meristem
cell differentiation. Thesedefects are at least in part caused by
effects on the BODENLOS/MONOPTEROS pathway and a resulting failure
in auxin signal-ing. As a consequence, expression of members of the
PIN andPLETHORA gene families is severely reduced in jlo
mutantroots (Bureau et al., 2010). During shoot development, JLO
isexpressed at sites of organ initiation and later in
meristem-to-organ boundaries. Ectopic high-level expression of JLO
in or-gan primordia causes leaf lobing and misexpression of bothSTM
and BP in developing organs. This indicates that JLOcould act from
the boundary to orchestrate gene expressionpatterns (Borghi et al.,
2007). However, all jlo mutant allelesdescribed so far grossly
disturbed embryogenesis and ar-rested growth already at early
stages, so that JLO functionsduring postembryonic development were
not yet understood.
Here, we characterized an allelic series of jlo alleles,
whichallowed us to uncover the role of JLO during organ
developmentin shoot and floral meristems. We find that JLO
integrates thepromotion of PIN transcription with the regulation of
KNOX ex-pression. We demonstrate that the JLO and AS2 proteins
in-teract molecularly and form multimeric complexes with AS1
tosuppress KNOX expression. Furthermore, we uncover a pre-viously
unsuspected role for AS2, together with JLO, in regu-lating auxin
transport in seedling roots.
RESULTS
Isolation of Novel jlo Alleles
The embryonic or early seedling lethality of jlo-1 and jlo-2
mu-tants interfered with any functional analysis during later
stagesof development (Borghi et al., 2007; Bureau et al., 2010).
Thus,
most of our conclusions regarding the function of JLO in
theshoot were drawn from misexpression experiments. We
nowcharacterized a series of novel jlo alleles that revealed
pheno-typically milder defects (jlo-3 to jlo-7, Figure 1A) and
allowed thedissection of JLO functions during later development.
RT-PCRanalyses of RNA isolated from seedlings showed that
insertionslocated 59 to the JLO transcriptional start (jlo-3 and
jlo-4) causea reduction in RNA levels, which are more severe in the
jlo-4allele. Homozygous jlo-5 to jlo-7 mutants produced
shortenedtranscripts that were truncated 39 to the insertions
(seeSupplemental Figure 1A online). Sequencing of theses
tran-scripts confirmed that the jlo-5 to jlo-7 alleles encode
proteinsthat lack parts of the conserved LOB domain (see
SupplementalFigures 1B and 1C online).Allelism tests were
subsequently performed. As expected for
allelic mutations, transheterozygosis of jlo-2 with jlo-3 to
jlo-7failed to complement the embryo mutant phenotypes and
seed-ling lethality of homozygous jlo-2 mutants (see
SupplementalTable 1 and Supplemental Figure 2 online). We conclude
that thephenotypic defects observed in the different jlo alleles
solely aredue to reduced or missing JLO function and not to
previouslyunnoticed mutations in other genes.
Developmental Defects in jlo-3 to jlo-7 Mutants
Plants homozygous for the jlo-4 mutation showed
phenotypicalterations from early stages of seedling development
onwards.Similar to the previously described jlo-2 allele (Bureau et
al.,2010), jlo-4 seedlings were smaller than wild-type seedlings
witha disorganized root and narrow cotyledons. However,
althoughgrowth and leaf development was strongly impaired,
homozy-gous jlo-4 mutants were eventually able to bolt (Figures 1D
to1D’’’). jlo-7 seedlings generated curled and fused leaves
(Fig-ures 1G and 1G’, arrowhead). The other jlo mutants studied
heredisplayed normal vegetative development.After the transition to
flowering, all novel jlo alleles displayed
related defects that were categorized into three classes. Class
Iincludes floral meristem identity defects (e.g., flower meristems
thatshow characteristics of inflorescence meristems) (Figure 1D’’’,
ar-rowhead). Some of these flowers were subtended by caulineleaves,
further supporting this assumption (Figure 1C’’, arrowhead).This
phenotype appeared with a low frequency (4.4%; n = 18/405)and
mainly within the first four flowers of jlo-3 and jlo-4 mutants(see
Supplemental Figure 3 and Supplemental Table 2 online).Class II
comprises homeotic transformations of petals and
stamen, which appeared mostly on the first flowers of jlo
mu-tants (25.4%; n = 103/405; Figure 1C’’’; see SupplementalFigure
3 and Supplemental Table 2 online). However, the ma-jority of
mutant flowers exhibited a reduced number and size ofsepals,
petals, and stamens or displayed organ fusions (55.8%;n = 226/405;
Figures 1E’’’ to 1G’’’). Together, these phenotypeswere classified
as class III.
The jlo Mutant Phenotype Is Dosage Dependent
jlo loss-of-function mutants arrest during embryogenesis orearly
seedling stages (jlo-1 and jlo-2), whereas reduced JLOactivity
causes leaf and floral defects (jlo-3 to jlo-7). In addition,
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we previously found altered target gene expression in
hetero-zygous jlo-2/+ plants (Bureau et al., 2010), suggesting that
plantdevelopment may be sensitive to the level of JLO activity.
Wetherefore compared shoot development of wild-type, jlo-2/+,and
jlo-2 plants to test this notion. Heterozygous
jlo-2/+mutantsappeared aphenotypic during vegetative development.
How-ever, after floral transition, jlo-2/+ flowers displayed
defects thatwere comparable to those of jlo-3 to jlo-7 mutants,
namely,homeotic transformations of second and third whorl
organs,
reduction in floral organ number, and organ fusions. Floral
budsopened prematurely due to smaller sepals and petals, and
sta-men size was notably reduced (see Supplemental Figure 4
on-line).jlo-2 homozygous mutants show a severe retardation in
shoot
growth. Scanning electron micrographs revealed that
mutantmeristems initiated primordia at arbitrary positions,
indicatingphyllotactic defects (Figures 2A to 2D). Most of these
organsfailed to grow out, and the remaining primordia gave rise
to
Figure 1. Analysis of jlo Mutant Alleles.
(A) Gene structure of JLO and the neighboring genes on
chromosome 4. The positions of the Ds element (jlo-2 to jlo-7) or
T-DNA (jlo-1) insertions areindicated. The jlo-1 and jlo-2 alleles
have been described previously. Black boxes, exons; white boxes,
untranslated regions; black arrows, start codon;asterisk, stop
codon.(B)/(B’) to (G)/(G’) Three-week-old wild-type plants (No-0)
compared with homozygous jlo-3 to jlo-7mutants and the
corresponding first four leaves. jlo-4 mutants are small and
develop misshapen leaves ([D]/[D’]). jlo-7 leaves curl upwards and
are occasionally fused ([G]/[G’]; arrowhead).(B’’) to (G’’) and
(B’’’) to (G’’’) Inflorescences and flowers of the wild type and
jlo mutants. Phenotypes comprised floral meristem identity defects
([C’’]and [D’’’] arrowheads), homeotic transformations ([C’’’];
arrowhead), reduced number of floral organs ([E’’’] and [F’’’]),
and organ fusions ([G’’’];arrowhead). Bars = 1 cm.[See online
article for color version of this figure.]
JLO and AS2 Regulate KNOX and PIN Genes 2919
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radialized organs (Figures 2B to 2D). By 25 d after
germination(DAG), the shoot meristem had stopped further growth.
Notably,this phenotype resembles that caused by inducible
misexpressionof a dominant-negative version of JLO (JLO-DN; Borghi
et al.,2007). Thus, both jlo-2/+ and jlo-2 plants exhibit defects
in organdevelopment, albeit with different severity.
Class I KNOX Genes Are Upregulated and EctopicallyExpressed in
jlo Mutants
Meristem development requires expression of class I KNOXgenes,
such as STM or BP, and their downregulation in lateralorgans. The
arrest of meristem activity in the jlo-2mutants couldbe caused by
changes in the activity or expression levels ofthese genes. Thus,
we examined BP and STM expression inhomozygous jlo-2
loss-of-function mutants. In the wild type,both BP:GUS (for
b-glucuronidase) and STM:GUS were ex-pressed only in meristematic
tissue and downregulated in organprimordia at 5 DAG (Figures 3A and
3C). By contrast, BP:GUSand STM:GUS signal intensity increased in
jlo-2 meristems andexpanded to the basis of lateral organ primordia
(Figures 3B and3D). At 15 DAG, BP and STM were expressed throughout
theenlarging apex and at the basis of organ primordia
(seeSupplemental Figure 5 online). Using quantitative RT-PCR
(qRT-PCR) assays, we found that transcript levels of BP and STM
areat least twofold increased in jlo-2 mutant seedlings.
Further-more, we observed an upregulation of both genes in jlo-2/+
andin jlo-4 to jlo-7 mutant seedlings (Figure 3G; see
Supplemental
Figure 1B online). To further test whether JLO regulates
organdevelopment via repression of BP and STM, we generated
thedouble mutants jlo-2 bp-1 and jlo-2 stm-2. As previously
pub-lished (Douglas et al., 2002), bp-1 mutants appear
aphenotypicduring vegetative development (Figure 3J), but produce
shorterinternodes and pedicels, together with downward-pointing
sili-ques after floral induction. In stm-2 single mutants, a
shootmeristem is initiated but arrested after generating a few
leaves(Figure 3L; Clark et al., 1996). When we combined jlo-2 with
bp-1or stm-2, primary leaves were visible at 14 DAG in jlo-2 bp-1
andjlo-2 stm-2 (Figures 3K and 3M) seedlings, before leaf
de-velopment and meristem activity was eventually arrested at
25DAG. By contrast, jlo-2 single mutants initiated only
radializedorgans (Figure 3I, inset), revealing that the reduction
in BP andSTM function in jlo-2 bp-1 and jlo-2 stm-2 double mutants
canpartially rescue the leaf growth defects of jlo-2 (see
SupplementalTable 3 online).
Genetic Interaction between JLO and AS2
Plants defective in AS2 function grow lobed leaves that
accu-mulate BP transcripts (Semiarti et al., 2001). Furthermore,
as2mutants develop flowers that open prematurely due to
reducedpetal and sepal sizes (Ori et al., 2000). Thus, jlo and as2
mutantsshare several characteristics. Because expression of both
genesoverlaps in newly initiated organ primordia, we
hypothesizedthat they might act in a common pathway to direct organ
de-velopment and regulate KNOX expression (Borghi et al.,
2007;Iwakawa et al., 2007; Soyano et al., 2008).We generated jlo-2
as2-1 and jlo-2 as2-2 double mutants to
analyze their genetic interactions. Both double mutant
combina-tions displayed similar genetic interactions (see below),
and onlythe jlo-2 as2-2 double mutants will be further discussed.
Com-pared with the wild type, jlo-2 embryogenesis is strongly
impairedwith aberrant patterning from the first cell division of
the pro-embryo onwards and an overall delay in development (Figures
4Kto 4M; Bureau et al., 2010), whereas as2-2 single mutants
areaphenotypic during embryo development (Figures 4F to 4H).
jlo-2as2-2 mutant embryos were indistinguishable from jlo-2
mutantembryos (Figures 4P to 4R), and the strong jlo-2 seedling
phe-notype was unaltered in jlo-2 as2-2 double mutants (Figures
4N,4O, 4S, and 4T). To analyze a genetic interaction during
laterstages of development, we also combined the as2-1 and the
as2-2mutation with the weaker jlo-3, jlo-5, and jlo-6 alleles
(Figure 4U).Again, the combination of jlo-3, jlo-5, and jlo-6 with
either as2-1 oras2-2 caused similar phenotypes, and only the jlo
as2-2 doublemutants will be further described. Compared with either
singlemutant, we observed increased leaf lobing and ectopic
leafletformation in all double mutant combinations, indicating
enhancedKNOX misexpression. Using qRT-PCR analysis (Figure 4V),
wefound a moderate increase of BP, but not of STM, transcript
levelsin jlo-3, jlo-5, and jlo-6 mutant leaves. As we did not
observe al-tered leaf morphology in the jlo-3, jlo-5, or jlo-6
single mutants(Figure 4U), the level of ectopic BP activity might
be too low toaffect leaf development. The expression of BP was
higher in as2-2mutant leaves and substantially higher in leaves of
each jlo as2-2double mutant combination. Notably, the ectopic
expression of BPin all jlo mutant leaves was not accompanied by any
reduction in
Figure 2. Shoot Development of jlo-2 Mutants.
Scanning electron micrographs of jlo-2 SAMs. Bars = 20 µm in (A)
and(B), 30 µm in (C), and 100 µm in (D).(A) Meristems reveal
phyllotactic defects at 5 DAG.(B) and (C) At 10 and 15 DAG, the
shoot apex is expanded and organsare initiated but fail to grow out
or appear radialized.(D) Some organs display leaf-like structures
at 25 DAG.
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AS2 transcripts. These results suggest that both JLO and AS2
areinvolved in the repression of BP during leaf morphogenesis.
We further analyzed flowers of jlo-2/+ as2-2 double mutants.With
respect to either single mutant, jlo-2/+ as2-2 sepals, petals,and
stamens were reduced in size, and cell length was de-creased (see
Supplemental Figure 4 online). Taken together,these genetic data
indicate that JLO and AS2 can functionpartially independently to
direct leaf and flower development.
AS2 Function Is Required for the JLOOverexpression Phenotype
The fact that STM and BP gene expression is upregulated in
jlomutant seedlings suggested that JLO normally acts to
down-regulate these two homeobox genes. However, this is in
con-trast with our previous observation that inducible
misexpressionof a fusion between JLO and the hormone binding domain
of theglucocorticoid receptor (GR) causes a drastic upregulation
ofSTM and BP expression (Borghi et al., 2007). To exclude
thatfusion to the GR domain interferes with the normal KNOX
re-pressing function of JLO, we designed an
estradiol-induciblei35S:JLO-FLAG transgene (Bureau et al., 2010).
JLO-FLAG–misexpressing plants revealed strongly lobed leaves,
resemblingthe JLO-GR misexpression phenotype (see Supplemental
Figure6F online) and showed enhanced transcript levels of BP andSTM
upon JLO-FLAG induction (see Supplemental Figures 6Gand 6H online).
Within 1 h after induction (HAI), both JLO proteinand RNA were
strongly upregulated (see Supplemental Figures
6I and 6J online). KNOX expression levels significantly
increasedwithin 4 HAI, suggesting an indirect mechanism of
upregulation(see Supplemental Figures 6G and 6H online). We
thereforespeculated that the transgenic high-level expression of
JLOmight interfere with the regulatory pathways that normally
re-strict KNOX expression. In line with this, we found that
JLOrequires AS2 for this activity because induction of JLO-FLAG
inas2-2 mutants did not alter the typical as2 leaf phenotype in96%
of all F2 plants analyzed (n = 53; see Supplemental Figure6D
online). Moreover, expression levels of BP and STM
remainedunaffected by JLO-FLAG expression in an as2-2 mutant
back-ground (see Supplemental Figures 6G and 6H online). From
thesedata, we conclude that JLO regulates KNOX expression
togetherwith AS2. Ectopic JLO expression could then either inhibit
AS2transcription or interfere with AS2-dependent regulation at
theprotein level. Because qRT-PCR analysis showed that AS2
tran-script levels are not altered in jlo mutants or upon JLO
mis-expression (Figures 3G and 4V; see Supplemental Figure 6K
online),we tested the possibility that both proteins physically
interact.
JLO and AS2 Can Physically Interact in Yeast
We performed GAL4-based yeast two-hybrid experiments toassay a
potential interaction between AS2 and JLO. AS2 wasfused to the GAL4
activation domain (GAL4-AD) and used asbait. Since full-length JLO
was unstable and the JLO C terminuswas activating transcription by
itself, we used only the N-terminalLOB domain fused to the GAL4 DNA
binding domain (GAL4-BD)
Figure 3. JLO Regulates Class I KNOX Gene Expression.
(A) to (F) BP:GUS ([A] and [B]), STM:GUS ([C] and [D]), and
AS2:GUS ([E] and [F]) expression in wild-type (WT) and
jlo-2mutants. In (B) and (D), BP andSTM expression is increased and
ectopic expression is detectable at the base of lateral organs
compared with (A) and (C). Arrowheads mark meristemboundaries.(G)
qRT-PCR of whole seedlings confirms reduction of JLO transcript
levels in jlo-2 mutants, while expression of BP and STM is
upregulated comparedwith the wild type (5 DAG). Note that gene
expression is already altered in jlo-2/+ seedlings. AS2
transcription is unaffected. MNE, mean normalizedexpression. Bars
indicate SE (n $ 3)(H) to (M) Genetic interactions between JLO and
the class I KNOX genes BP and STM. Pictures of the wild type (H),
jlo-2 (I), bp-1 (J), jlo-2 bp-1 (K), stm-2(L), and jlo-2 stm-2 (M)
mutants were taken 14 DAG. Homozygous jlo-2 initiate radialized
organs (arrowhead; inset shows a close-up; [I]). bp-1
mutantsexhibit a wild type–like appearance (J), while stm-2 mutants
initiate a SAM, which arrests growth after initiation of a few
leaves (L). jlo-2 bp-1 (K) and jlo-2stm-2 (M) double mutants show
partial rescue of the jlo-2 phenotype and produce primary leaves
before the meristem finally arrests activity.Bars = 50 mm in (A) to
(F), 50 mm in (H) and (J), and 10 mm in (I) and (K) to (M).[See
online article for color version of this figure.]
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as prey. In this assay, AS2 was able to interact with the JLO
LOBdomain, as demonstrated by growth on selective medium
lackingLeu, Trp, His, and Ade (Figure 5B).
To map the domains relevant for the interaction, we
testedseveral truncations of both proteins. Interaction was
observedwhen AS2 was combined with JLO versions carrying the
GASBLOCK and coiled coil domain, which are highly conservedamino
acid regions within the LOB domain (Figure 5B; Shuaiet al., 2002).
None of the AS2 truncations, including only parts orthe complete
LOB domain, were able to interact with JLO. Thissuggests that
domains within the AS2 C-terminal region mediatethe interaction.
However, the AS2 C terminus fused to theGAL4-AD appeared to be
toxic for yeast (Figure 5B). Thus, weperformed yeast two-hybrid
experiments with AS2 GAL4-BD fu-sions as prey (Figure 5C).
Interaction with JLO was obtained with
full-length AS2 or only the C-terminal domain of AS2. In
yeast,both JLO and AS2 can also interact with LBD31, an LBD
proteinclosely related to JLO, opening up the possibility that LBD
pro-teins can form higher order complexes and act in a
combinatorialfashion. However, the interactions are not random
between LBDproteins, as, for instance, LBD2 was not able to bind
JLO or AS2in yeast assays (see Supplemental Figure 7 online).
JLO Interaction with AS1 Is Mediated by AS2
Complex formation between AS2 and AS1 was shown to berequired
for BP repression in lateral organs (Xu et al., 2003; Guoet al.,
2008). Coexpression of AS2 and AS1 allowed yeastgrowth on selective
media, thus verifying the previously pub-lished data. However, we
did not observe any direct interaction
Figure 4. Genetic Interaction of JLO and AS2.
(A) to (T) Embryonic and seedling development (5 DAG) of the
wild type (WT) ([A] to [E]), as2-2 ([F] to [J]), jlo-2 ([K] to
[O]), and jlo-2 as2-2 mutants ([P]to [T]). Embryonic stages:
16-cell stage ([A], [F], [K], and [P]), heart stage ([B], [G], [L],
and [Q]), and torpedo stage ([C], [H], [M], and [R]). as2-2embryos
are aphenotypic; jlo-2 and jlo-2 as2-2 embryos reveal altered cell
division planes at the early proembryo stage ([K] and [P]). These
embryos didnot develop beyond the heart stage. Insets in (A), (F),
(K), and (P) show the respective embryo schematically. From the
heart stage onwards, jlo-2 andjlo-2 as2-2 embryos displayed reduced
hypocotyl diameter and length and showed an overall developmental
delay. Postembryonically, as2-2 mutantsdeveloped lobed leaves (I)
but normal roots (cf. [E] and [J]). jlo-2 and jlo-2 as2-2 seedlings
developed asymmetric and atrophic cotyledons, shorthypocotyls, and
a defective root organization ([N] and [S], and [O] and [T]). Roots
were stained via the modified pseudo-Schiff propidium iodidemethod;
black dots indicate the starch granules in differentiated columella
cells.(U) Silhouettes of mature leaves of the wild type (No-0),
jlo-3, jlo-5, jlo-6, and as2-2 single mutants as well as jlo-3
as2-2, jlo-5 as2-2, and jlo-6 as2-2double mutants.(V) qRT-PCR
analysis of BP, STM, and AS2 expression in mature leaves. BP
expression is elevated in jlo-3, jlo-5, and jlo-6 leaves, further
upregulated inas2-2 leaves, and increased even more in each jlo
as2-2 double mutant combination. No change in STM transcript level
is detectable in mature leaves ofeach mutant background. AS2
expression remains unaltered in jlo-3, -5, and -6 mutant leaves but
is strongly decreased in as2-2 single and jlo as2-2double mutant
leaves. MNE, mean normalized expression. Bars indicate SE (n $
3).Bars = 20 µm in (A), (F), (K), and (P), 50 µm in (B), (C), (E),
(G), (H), (J), (L), (M), (O), (Q), (R), and (T), 1 mm in (D), (I),
(N), and (S), and 0.5 cm in (U).[See online article for color
version of this figure.]
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between JLO and AS1 (Figure 6). We therefore performed a
yeastthree-hybrid assay to test whether JLO, AS2, and AS1 have
thepotential to form a multimeric complex. Yeast was able to grow
onselective medium when all three proteins were expressed, show-ing
that JLO can interact indirectly with AS1 through AS2.
Theobservation of such higher order complexes between JLO, AS2,and
AS1 could help explain the effects of JLO misexpression onKNOX
expression (see Supplemental Figure 6F online). Here, highlevel
expression of JLO in a wild-type background may interferewith the
KNOX-restricting activity of the AS1/AS2 complex, pos-sibly by
sequestering AS2 into JLO/AS2 or multimeric complexes.
Intracellular Localization of Fluorescent Protein–TaggedJLO,
AS2, and AS1
To analyze protein interaction in planta, JLO, AS2, and AS1
werefused to the fluorescent proteins (FPs) GFP or mCherry
andtransiently expressed in Nicotiana benthamiana leaf
epidermal
cells. We used a b-estradiol expression system to limit
over-expression artifacts and unspecific interactions (Bleckmannet
al., 2010). Integrity of the different fusion proteins was
con-firmed by immunoblotting using an anti-GFP antibody
(seeSupplemental Figure 8G online). JLO and AS2 fusion proteinswere
found in the cytoplasm and enriched in the nucleoplasm(Figures 7A
and 7A’). AS1 was localized to the nucleus withhigher protein
abundance in the nucleolus (Figure 7B’). Con-sistent with
previously published results, the presence of AS2caused
relocalization of AS1 to the nucleoplasm (Zhu et al.,2008; Figures
7C and 7C’’). By contrast, JLO did not affect AS1localization,
supporting the notion that JLO and AS1 do notdirectly interact
(Figures 7B and 7B’’). We used inducible mis-expression in stably
transformed Arabidopsis plants to test thefusion proteins for
functionality. In all cases, we obtained thepreviously described
gain-of-function phenotypes (Iwakawaet al., 2002; Xu et al., 2003;
Borghi et al., 2007; Zhu et al., 2008),indicating that the fusion
proteins are fully active. The observed
Figure 5. Mapping the JLO–AS2 Interaction Domains.
(A) Protein structure of JLO and AS2. The LOB domain at the N
terminus of each protein consists of a C-BLOCK (turquoise/orange),
GAS-BLOCK(purple/green), and coiled coil (CC; gray/black)
domain.(B) and (C) GAL4-based yeast two-hybrid study. Mating with
empty pGADT7 and pGBKT7 vectors excludes autoactivation. Growth on
-Leu/Trp mediawas used to select for both plasmids.(B) Growth on
selective media (-Leu/Trp/His/Ade) was only detected for JLO
versions, including the GAS-BLOCK and coiled coil domain. The AS2
Cterminus fused to Gal4-AD appeared to be toxic.(C) AS2 full-length
or a C-terminal truncation was able to interact with JLO. All
results were verified via calculation of Miller units in a liquid
culture assay(black bars). Mating with pGADT7 (white bars) and
pGBKT7 (gray bars) was used to calculate the background (gray
shadowed). Asterisks indicatea significant difference to background
(P $ 0.05; analyzed by Student’s t test). Bars indicate SE (n $
3).[See online article for color version of this figure.]
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subcellular localizations in Arabidopsis root epidermal
cells(Figures 7D to 7F) resembled those in N. benthamiana
leafepidermal cells (Figures 7A to 7C). Similarly, coexpression
ofAS2/AS1 (Figures 7F and 7F’’) but not of JLO/AS1 (Figures 7Eand
7E’’) resulted in a relocalization of AS1 to the nucleoplasm.These
findings are again consistent with a direct interactionof JLO/AS2
and AS2/AS1 and no direct binding of JLO to AS1.
Fluorescence Resonance Energy Transfer–Based ProteinInteraction
Analysis Supports the Formation of JLO/AS2/AS1 Complexes
Next, we measured fluorescence resonance energy transfer
ef-ficiencies (EFRET) between the GFP and mCherry pairs
(Albertazziet al., 2009) in planta. EFRET was calculated as the
percentageincrease of GFP (donor) fluorescence after photobleaching
ofmCherry (acceptor) (Bleckmann et al., 2010). All
photobleachingexperiments and EFRET measurements were performed in
the
nucleus. In N. benthamiana leaf epidermal cells,
fluorescentsignals were first detectable at 1 HAI and remained
stable over12 h (see Supplemental Figure 8E online). Upon extended
in-duction ($24 HAI), some cells carried fluorescent
aggregates,indicating protein overexpression (see Supplemental
Figure 8Fonline, arrowhead). Therefore, all measurements were
per-formed within 12 HAI. As EFRET depends on the orientation
anddistance of both chromophores to each other, we
measuredintramolecular EFRET as a control for the minimal distance.
Tothis end, both GFP and mCherry were fused together to the
Ctermini of all tested proteins. The intramolecular EFRET
wemeasured for all fusion proteins ranged from 26 to 28%.
Cal-culation of GFP fluorescence fluctuation during
photobleachingin the absence of the donor revealed a maximal
background of6% in all control experiments. Thus, only EFRET
significantly higherthan 6% was regarded as an indication of close
proximity orphysical interaction of the two proteins for this set
of experiments(Figure 7G).
Figure 6. Yeast Three-Hybrid Assay.
(A) Protein structure of JLO, AS2, and AS1. JLO and AS2: C-BLOCK
(turquoise/orange), GAS-BLOCK (purple/green), and coiled coil
domain (CC; gray/black). AS1: MYB domain (blue) and coiled coil
domain (red).(B) GAL4-based yeast studies revealed an interaction
between AS2 and AS1 but not between JLO and AS1. A yeast
three-hybrid assay showed thatAS2 can bridge the interaction
between JLO and AS1. AS2 was cloned in the pTFT1 vector and
cotransformed with AS1-GAL4-AD and JLO(LOB)-GAL4-BD vectors into
yeast. Growth on -Leu/Trp media was used to select for GAL4-AD and
GAL4-BD constructs. Growth on selective media (-Leu/Trp/His/Ade)
was used to monitor interactions. Cotransformation with empty
pGADT7 and pGBKT7 vectors exclude autoactivation.(C) All results
were verified via calculation of Miller units in a liquid culture
assay (black bars). Cotransformation with pGADT7 (white bars) and
pGBKT7(gray bars) was used to calculate the background (gray
shadowed). Asterisks indicate a significant difference to
background (P $ 0.05, analyzed byStudent’s t test). Bars indicate
SE (n $ 3).[See online article for color version of this
figure.]
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The results we obtained confirmed our yeast GAL4
interactionstudies and showed a clear JLO/AS2 (EFRET = 15% 6 0.5%)
andAS2/AS1 (EFRET = 23% 6 2%) interaction in both reciprocal
GFP/mCherry combinations. By contrast, we did not observe a
signif-icant EFRET for JLO/AS1 (4.3% 6 0.8%). However, when
wecoexpressed untagged AS2, significant EFRET between JLO andAS1
was recorded (14.5%6 1%). Measurements performed at 1,2, 4, and 12
HAI revealed that EFRET remained stable over time(see Supplemental
Figure 8H online), indicating that EFRET asa measure of protein
interaction did not depend on proteinoverexpression. Interestingly,
JLO (EFRET = 11% 6 1.0%), AS2(EFRET = 17% 6 1.7%), and AS1 (EFRET =
8.9% 6 1.5%) showedsignificant homomerization (Figure 7G),
suggesting that varioushomomers and heteromers may coexist within
the nucleus.
FP-tagged LBD31 and LBD2 proteins were used to furthertest the
specificity of the observed interactions. Both proteinslocalized to
the nucleus and cytoplasm, thus resembling sub-cellular
localizations of JLO and AS2 (see Supplemental Figures8A to 8D
online). In line with our yeast studies, we detected aninteraction
between JLO/LBD31 (11.1% 6 1.0%) and AS2/LBD31 (15.0% 6 1.9%),
whereas EFRET values for JLO/LBD2(6.8% 6 0.7%) and AS2/LBD2 (7.1% 6
1.0%) were close tobackground (Figure 7G).
Overall, we noted a reduction of EFRET in root epidermal cellsof
stably transformed Arabidopsis plants compared with
N. benthamiana (Figure 7H). The intramolecular EFRET we
mea-sured ranged only from 15 to 17%, with a GFP
backgroundfluctuation of 1.5%. We verified heteromerization of
JLO/AS2(4.1% 6 0.2%) and AS2/AS1 (8.6% 6 0.4%) as well as
homo-merization of JLO (3.1 6 0.3%), AS2 (4.4 6 0.3%), and AS1(2.4
6 0.1%). EFRET values for JLO/AS1 (1.6% 6 0.4%) were inthe range of
background level.We conclude that JLO and AS2 specifically interact
in yeast,
N. benthamiana, and Arabidopsis.
AS2 and JLO Regulate Auxin Transport
Our studies revealed the capacity of JLO and AS2 to formprotein
complexes in Arabidopsis and that both proteins can acttogether in
the regulation of KNOX expression in the shoot. JLOwas previously
shown to regulate auxin transport and signalingfrom embryogenesis
onwards, and we therefore asked if JLOand AS2 share functions
during early stages of development.Expression of AS2 was analyzed
using an AS2:GUS reportergene construct (Jun et al., 2010). GUS
signals appeared on theadaxial side of cotyledons and organ
primordia during embry-onic and postembryonic development. In
addition, AS2:GUSwas expressed in cells of the suspensor and the
embryonic roottip (Figures 8F to 8H). After germination, AS2 is
expressed in thetips of seedling roots (Figure 8I). JLO expression
in embryos was
Figure 7. Intracellular Protein Localization and FRET-Based
Protein Interaction Analysis.
(A) to (C) Colocalization of FP-tagged proteins in N.
benthamiana epidermis cells: colocalization of JLO-GFP and
AS2-mCherry ([A] to [A"]), colo-calization of JLO-GFP and
AS1-mCherry ([B] to [B"]), and colocalization of AS2-GFP and
AS1-mCherry ([C] to [C"]). Bars in (A) and (B) indicate SE.(D) to
(F) Colocalization of FP-tagged proteins in Arabidopsis root
epidermis cells: colocalization of JLO-GFP and AS2-mCherry ([D] to
[D"]), colo-calization of JLO-GFP and AS1-mCherry ([E] to [E"]),
and colocalization of AS2-GFP and AS1-mCherry ([F] to [F"]). Bars =
10 mm in (A) to (F).(G) and (H) Protein interaction revealed by
percent EFRET. Intramolecular EFRET obtained by direct fusion of
GFP to mCherry in a single molecule (graybars) and GFP background
fluctuation (white bars) were calculated as positive and negative
controls. Control measurements were performed for allproteins
tested and were similar in all experiments. Gray shaded area
indicates background fluctuation level of GFP. Asterisks mark a
significantdifference from background (*P # 0.05 and **P # 0.01;
analyzed by Student’s t test).(G) EFRET measured with a transient
expression of FP-tagged protein in epidermis cells of N.
benthamiana (n$ 35 for each combination) AS2 (red): EFRETmeasured
with coexpression of FP-tagged JLO and AS1 together with untagged
AS2 protein.(H) EFRET measured in root epidermis cells of stable
transformed Arabidopsis plants (n $ 25 for each combination).[See
online article for color version of this figure.]
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Figure 8. Gene Expression Analyses during Embryonic and Root
Development.
Embryonic stages: heart stage ([A], [F], and [K] to [P]),
torpedo stage ([B] and [G]), and bent cotyledon stage ([C] and
[H]).(A) to (C) Embryonic expression pattern of JLO analyzed by
whole-mount RNA in situ hybridization.(A) At the heart stage, JLO
transcripts are detectable in provascular cells. WT, wild type.(B)
and (C) Later on, expression becomes restricted to the basal root
tip ([C]; arrowhead) and the stele.(D) and (E) Expression of a
JLO:GUS reporter in wild-type seedlings (5 DAG). JLO is expressed
in the stele as well as in columella cells of primary roots(D). In
mature leaves, the JLO:GUS reporter is strongly expressed in the
vascular system and hydathodes. Weaker expression is also
detectable in theleaf blade (E).(F) to (H) During embryogenesis,
AS2:GUS is expressed at the adaxial side of cotyledons and the
basal root tip (arrowhead in [H]), including thesuspensor.(I) AS2
expression at the tip of wild-type roots.(J) jlo-2 roots reveal an
unaltered AS2 expression.(K) to (M) DR5rev:GFP expression in heart
stage embryos.(K) In the wild type, auxin concentrates in the root
primordium and suspensor as well as in the tips of the
cotyledons.(L) jlo-2 embryos reveal only a weak signal at the root
pole.(M) as2-2 embryos show unaltered DR5rev activity.(N) to (P)
PIN1:PIN1-GFP expression in heart stage embryos.(N) PIN1
concentrates to the cotyledon tips and provascular cells in the
wild type.(O) and (P) jlo-2 (O) and as2-2 (P) mutants show similar
PIN1 distributions but reduced expression levels.(Q) qRT-PCR
analysis of JLO and AS2 transcript levels (5 DAG). JLO expression
is reduced in jlo-2 mutants but unaltered in the as2-2 mutant
background.Similarly, AS2 expression is downregulated in
as2-2mutants but unchanged in jlo-2 seedlings. MNE, mean normalized
expression. Bars indicate SE (n$ 3).Bars = 50 mm.[See online
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analyzed through whole-mount RNA in situ hybridization. At
theearly heart stage, JLO is expressed in the entire embryo witha
stronger transcript accumulation in the basal domain (Figure8A).
Later on, expression is confined to the embryo axis, thedeveloping
vasculature, and the root pole (Figures 8B and 8C).Thus, the JLO
and AS2 expression domains overlap at the heartstage and in the
basal root tip of later embryo stages. The ex-pression pattern of
JLO during postembryonic development wasanalyzed using a JLO:GUS
reporter construct. In the shoot, JLOis expressed in the
vasculature of cotyledons and leaves, andweaker signals were also
detected in the leaf blade (Figure 8E).In seedling roots, JLO:GUS
is strongly expressed in the steleand in the root tip, including
the columella cells (Figure 8D), thusoverlapping extensively with
the expression of AS2 in thesetissues. Notably, the JLO expression
levels are unaltered in as2-2 mutants (Figure 8Q). Similarly,
neither expression domain norexpression levels of AS2 are altered
in the jlo-2 mutant back-ground (Figures 8J and 8Q).
The severe patterning defects of jlo-2 embryos and roots re-sult
from a failure in auxin signaling and transport (Bureau et
al.,2010). Thus, we examined auxin distribution in as2-2
mutantsusing the synthetic auxin response reporter
DR5rev:GFP(Ulmasov et al., 1997). In wild-type heart stage embryos,
auxinaccumulates at the root pole with an intensity maximum in
theuppermost suspensor cell (Figure 8K). Unlike jlo-2 mutants,
as2-2 embryos displayed an unaltered DR5rev:GFP activity (Figures8L
and 8M). Postembryonic roots exhibit a maximum of DR5rev:GFP
expression in the quiescent center, the adjacent stem cells,and
columella cells. Again, DR5rev activity was unaltered inseedling
roots of as2-2 mutants (Figures 9A and 9E). Becauseauxin
accumulation is regulated by several PIN proteins, weanalyzed their
expression in more detail (Vieten et al., 2005). Wepreviously
identified JLO as an important regulator of these ef-flux carriers
(Bureau et al., 2010). Consistent with this, expres-sion levels of
PIN1/3/4 and PIN7 were reduced in the novel jlo-3to jlo-7 alleles
(see Supplemental Figure 1B online). We alsoanalyzed the expression
of PIN1:PIN1-GFP, PIN4:PIN4-GFP,and PIN7:PIN7-GFP reporters in the
as2-2 mutant background.We found a decreased PIN1-GFP signal in
as2-2 embryos androots (Figures 8P and 9F). PIN7-GFP expression in
as2-2 rootswas also reduced, whereas PIN4-GFP signals were
stronglyincreased (Figures 9G and 9H). Thus, AS2 and JLO are
similarlyrequired for PIN1 and PIN7 expression, but they differ in
theircapacity to regulate PIN4 expression.
The JLO-AS2 Complex Regulates PIN Expression
We sought to analyze whether JLO and AS2 function together inthe
transcriptional regulation of PIN1,3,4 and 7. To this end, wefirst
performed qRT-PCR analysis on jlo-2 and as2-2 single anddouble
mutant roots (Q). PIN1 and PIN3 transcript levels arereduced in
jlo-2 mutants, and we found a similar downregulationof these genes
in the as2-2 mutant background. jlo-2 as2-2double mutants revealed
no further decrease in PIN1 and PIN3transcription, suggesting that
JLO and AS2 act together topromote PIN1 and PIN3 expression.
However, PIN4 expressionwas reduced in jlo-2 mutants but nearly
threefold upregulated inas2-2 mutants. In jlo-2 as2-2 double
mutants, PIN4 expression
levels resembled those of jlo-2 single mutants. This
indicatesthat JLO is essential for PIN4 expression and that
JLO/AS2heteromers may act to restrict PIN4 levels. PIN7
transcription isnotably reduced in both jlo-2 and as2-2 mutant
roots but furtherdecreased in the double mutant, indicating that
JLO acts par-tially independently of AS2 to promote PIN7
expression.To further identify AS2-dependent functions of JLO, we
ana-
lyzed the expression of the DR5rev:GFP and PIN:PIN-GFP re-porter
in roots (5 DAG) upon JLO-FLAG induction (12 HAI).Increased JLO
activity was able to upregulate DR5rev:GFP andPIN1:PIN1-GFP but not
PIN7:PIN7-GFP expression in wild-typeroots, while PIN4-GFP signals
decreased within 12 HAI of JLO(Figures 9I to 9L). In the as2-2
mutant background, DR5rev:GFPand PIN1:PIN1-GFP expression did not
respond to JLO-FLAGinduction, suggesting that JLO requires AS2 for
this function(Figures 9M and 9N). Expression of PIN4-GFP, which is
alreadyincreased in as2-2 mutant roots compared with the wild
type,remained unaltered upon JLO-FLAG induction (Figure
9O).Similarly, PIN7-GFP signals did not respond to increased
JLOexpression (Figure 9P).Using qRT-PCR assays, we confirmed that
increased JLO
activity is sufficient for PIN1 as well as PIN3 but not for
PIN7upregulation in wild-type roots. Expression of PIN4 was
firstinsensitive to JLO induction and decreased after prolonged
(12HAI) JLO induction (Figure 9R; Bureau et al., 2010). In
as2-2mutants, PIN1, PIN3, and PIN7 transcript levels did not
respondto JLO-FLAG induction, while JLO-FLAG expression causeda
minor increase in PIN4 RNA levels (Figure 9S). Thus, both JLOand
AS2 are required for positive regulation of PIN1, 3, and
7expression. JLO also contributes to complexes that promotePIN4
expression, which may compete with JLO/AS2 in theregulation of
PIN4. This would imply that correct PIN4 expres-sion requires a
precise balance of various complexes that in-volve JLO.
Interference via JLO misexpression may increaseJLO/AS2
dimerization, thus repressing PIN4, while the absenceof AS2 in
as2-2 mutants would preferentially allow the formationof
PIN4-promoting complexes (Figure 10).
DISCUSSION
Organ initiation requires the accumulation of auxin and the
si-multaneous downregulation of class I KNOX expression in
organanlagen, as well as the establishment of boundaries that
separatecells with different cell fates (reviewed in Rast and
Simon, 2008).Several lines of evidence suggest that JLO plays an
essential roleduring cell fate regulation. (1) JLO is expressed
early at sites oforgan initiation, in meristem-to-organ boundaries,
and in matureleaves (Borghi et al., 2007; Soyano et al., 2008). (2)
CompromisedJLO activity causes pleiotropic defects that can be
classified toaffect meristem activity, organ identity, growth, and
separation. (3)These defects are accompanied by ectopic expression
of STMand BP and misregulated expression of several PIN genes.We
now conclude that JLO function is required for the repres-
sion of class I KNOX genes during lateral organ
developmentbecause STM and BP expression were expanded into the
basaldomain of organ primordia in jlo-2 mutants. This KNOX
genemisexpression is, at least in part, responsible for organ
de-velopmental defects in jlo-2 mutant meristems as we observed
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Figure 9. PIN Expression Is Regulated by a JLO/AS2 Complex.
(A) to (P) Expression of DR5rev:GFP ([A], [E], [I], and [M]),
PIN1:PIN1-GFP ([B], [F], [J], and [N]), PIN4:PIN4-GFP ([C], [G],
[K], and [O]), and PIN7:PIN7-GFP ([D], [H], [L], and [P]) in the
wild type ([A] to [D]) and as2-2 ([E] to [H]) mutants and after
induced expression of JLO-FLAG in the wild type ([I] to [L])and
as2-2 mutant background ([M] to [P]) (5 DAG). Auxin accumulation is
unaltered in as2-2 mutants (E). PIN1-GFP and PIN7-GFP signals are
severelyreduced ([F] and [H]), while PIN4-GFP signal is strongly
increased (G) in as2-2 roots. Col-0, Columbia-0. Bars = 50 mm.(I)
to (L) Compared with the wild type ([A] and [B]), DR5rev promoter
activity and PIN1-GFP expression is upregulated upon JLO-FLAG
induction (12HAI), while PIN4-GFP signal is reduced (K) and the
PIN7-GFP signal remains unaltered (L).(M) to (P) In as2-2mutants,
induction of JLO-FLAG expression does not alter DR5rev:GFP (cf. [E]
and [M]) expression. Similarly, PIN1:PIN1-GFP (N), PIN4:PIN4-GFP
(O), and PIN7:PIN7-GFP (P) expression remains unaffected by high
level JLO misexpression in as2-2 mutant background (cf. with [F] to
[H]).(Q) qRT-PCR analysis reveals a downregulation of PIN1, PIN3,
and PIN7 and an upregulation of PIN4 in as2-2 roots. By contrast,
transcript levels of allPIN genes studied are strongly reduced in
jlo-2 and jlo-2 as2-2 roots. Note that PIN1, PIN3, and PIN4
expression is similar in jlo-2 and jlo-2 as2-2mutants, while PIN7
transcript levels are further reduced in the double mutant. MNE,
mean normalized expression.(R) and (S) Analysis of PIN1, PIN3,
PIN4, and PIN7 transcript levels in roots after induced
misexpression of JLO-FLAG in the wild type (R) and as2-2mutants
(S). Expression levels were normalized to uninduced controls
prepared at the same time points. In the wild type (R), PIN1 and
PIN3 areupregulated within 12 HAI, while PIN4 is downregulated and
PIN7 expression remained unaltered. No significant response of
PIN1, PIN3, and PIN7expression is detectable in as2-2 mutants upon
JLO-FLAG induction, while PIN4 expression is slightly upregulated.
Bars in (Q) to (S) indicate SE (n$ 3).[See online article for color
version of this figure.]
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a partial rescue of the phenotype in jlo-2 bp-1 and jlo-2
stm-2double mutants. The elevated levels of BP in mature leaves
ofthe weaker jlo-3, jlo-5, and jlo-6 alleles, which still permit
organformation, most likely due to residual JLO activity, further
in-dicate a role for JLO in the maintenance of BP repression.
JLOshares this role with AS2, which interacts with the MYB
proteinAS1 to repress BP expression during leaf morphogenesis
(Oriet al., 2000; Xu et al., 2003; Guo et al., 2008).
Our studies showed that JLO and AS2 can physically interactin
vivo. Furthermore, we found that AS2 can mediate the in-teraction
between JLO and AS1. Therefore, a simple scenariocould be that a
JLO/AS2/AS1 complex represses BP expressionin developing organs.
However, the synergistic effects in jlo-3,-5,-6 as2-2 and jlo-2/+
as2-2 mutants indicate that JLO can actalso independently of AS2.
Furthermore, STM was found to bemisexpressed in jlo mutant
backgrounds but not in as2-2 mu-tants (Ori et al., 2000). This
suggests that both JLO/AS2/AS1heteromeric complexes as well as JLO
homomers may regulateKNOX expression in the shoot (Figure 10).
The evidence for JLO acting as a negative regulator of BP andSTM
expression is entirely based on the loss-of-function data andthus
likely reflects genuine JLO activity. We found that JLO acquiresthe
capacity to interfere with KNOX repression when overexpressed.The
simplest explanation for this observation is that increased
JLOexpression disturbs the regulatory networks that normally serve
torestrict KNOX expression from leaves. Since organ development
isalso highly sensitive to reduced JLO dosage, we conclude that
JLOexpression levels must be precisely regulated.
Our misexpression experiments showed that JLO induction inthe
as2-2 mutant background did not affect BP and STM tran-script level
or enhance the as2-2 leaf phenotype, suggestingthat JLO acts with
or through AS2. However, we previouslynoted that JLO overexpression
phenotypes do not depend onAS1 activity (Borghi et al., 2007). We
therefore propose thata high level JLO expression interferes with
an AS1-independent
function of AS2. Such independent activities have been
pre-viously suggested because AS1 and AS2 are expressed inlargely
overlapping but not identical expression patterns duringlateral
organ development, and AS2 overexpression phenotypesstrongly differ
from those caused by AS1 misexpression (Byrneet al., 2002; Iwakawa
et al., 2002, 2007). Thus, JLO and AS2 mayalso form complexes that
do not include AS1 but serve to reg-ulate KNOX expression (Figure
10).Our studies of JLO and AS2 function during root development
further support the hypothesis that both proteins act together
inheteromeric complexes. Mutant studies showed that JLO andAS2 act
together to promote PIN1 and PIN3 expression and thatfurther PIN1
and PIN3 upregulation by JLO overexpression re-quires the presence
of AS2. We found increased PIN4 expressionin as2-2 single mutants
but a decrease in jlo-2 or jlo-2 as2-2double mutants. A simple
interpretation of these results is thatJLO homomers strongly
promote PIN4 transcription, JLO/AS2heteromers regulate normal PIN4
levels, and AS2 homomers (orcomplexes involving also AS1) repress
PIN4 expression (Figure10). This notion is supported by the
observation that JLO in-duction in the absence of AS2 further
increased PIN4 transcrip-tion. To this end, it is noteworthy that
PIN proteins are to someextent functionally interchangeable and
that loss of PIN expres-sion can be compensated for by other PIN
genes (Blilou et al.,2005; Vieten et al., 2005). Thus, PIN4
expression may be en-hanced by JLO homomers in as2-2 mutants, which
may com-pensate for the reduced expression of PIN1, PIN3, and
PIN7.Finally, based on our jlo-2 as2-2 double mutant analysis, JLO
andAS2 act at least partially independently to direct PIN7
expression.We reported previously that PIN expression is already
re-
duced at the early stages of jlo embryogenesis (Bureau et
al.,2010). Similarly, we showed here reduced PIN1 expression
inas2-2 heart stage embryos. Thus, JLO and AS2 complexes
likelyregulate PIN expression from embryogenesis onwards. We
alsonoted that the SAM of jlo-2 mutants initiated filamentous
organswith a disturbed phyllotaxis and eventually arrested organ
for-mation. Mutations in PIN1 also cause the formation of
radializedorgans and an arrest of lateral organ initiation from the
shootmeristem (Vernoux et al., 2000; Reinhardt et al., 2003).
There-fore, the overall reduction in PIN1 transcription, which
weshowed for jlo-2 mutant seedlings (Bureau et al., 2010),
likelyalso contributes to the aberrant jlo-2 shoot development.
Thisassumption is consistent with the observation that mutations
inBP or STM are insufficient to fully restore the meristem
activityof homozygous jlo-2 seedlings in the double mutant
combina-tions. Taken together, the epistatic genetic interaction
betweenjlo-2 and as2-2 at the embryonic and seedling stages,
combinedwith our analysis of root development, suggests a
continuousrequirement for JLO homomeric and JLO/AS2
heteromericcomplexes throughout plant development.Our results show
that JLO and AS2 regulate a similar set of
target genes and act in a combinatorial manner. This raises
thequestion of how the JLO/AS2 complexes are modulated toobtain
their specific functions. A possible explanation is that
theinteraction with additional factors is responsible for the
modu-lation of DNA binding specificity or transcriptional
regulation.Similar combinatorial activities were reported for MADS
boxproteins, which determine the identity of floral organs
(Theissen
Figure 10. A Model for KNOX and PIN Regulation through
Homomericand Heteromeric JLO-AS2 Complexes.
Complexes containing JLO and AS2, either JLO/AS2/AS1 trimeric
orJLO/AS2 heteromeric complexes, repress BP and PIN4 expression
andpromote PIN1, PIN3, and potentially PIN7 expression. The
interactionbetween JLO and AS1 is mediated through AS2. JLO
homomers repressSTM and BP expression and promote PIN4 and PIN7
expression. AS2homomeric complexes may promote PIN7 expression
independently.Correct target gene expression requires a tight
balance between thevarious protein complexes. LBD31 can bind to JLO
and AS2, but theregulatory function of these complexes is so far
unknown.[See online article for color version of this figure.]
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and Saedler, 2001). The formation of the multimeric
JLO/AS2/AS1complex in yeast supports this assumption. AS1 is
expressed indeveloping organ primordia, leaves, and roots (Iwakawa
et al.,2007). Thus, JLO/AS2 could function with AS1 to regulate
KNOXand PIN expression in both shoots and roots. Indeed, the as2
andas1 mutant leaf phenotypes were not fully suppressed by
bpmutants, suggesting that both genes regulate other targets
be-sides BP (Ori et al., 2000; Byrne et al., 2002). In addition,
auxinactivity is asymmetrically distributed at the distal leaf tip
of as1 oras2 mutants (Zgurski et al., 2005). These observations
could beexplained by postulating a role for AS2 and AS1 in PIN
regulation,which is strongly supported by our data presented here.
In theroot system, where no as1 mutant phenotype was described
sofar, other MYB genes may replace AS1 function and contribute
toPIN regulation.
We found that both JLO and AS2 can interact also with LBD31in
vivo. LBD31 expression is first detectable at sites of
organinitiation and later in meristem-to-organ boundaries. This
ex-pression pattern overlaps with that of JLO, AS2, and AS1
duringearly organ development and later with that of JLO in
theboundary (Borghi et al., 2007; Iwakawa et al., 2007; Xu et
al.,2008; Jun et al., 2010). Whether higher order complexes
withLBD31 are formed has not been analyzed so far. However,
weenvisage that LBD proteins can undergo a wide range of
com-binatorial interactions with each other. In contrast with jlo
or as2and as1 mutants, knockout alleles of LBD31 were
aphenotypic,indicating that either JLO can compensate for the loss
of its closehomolog LBD31 or that other related LBD proteins can
substitutefor LBD31 in heteromeric complexes. Further experiments
arerequired to distinguish between these possibilities.
Reduced JLO activity interfered with the establishment
ofboundaries, resulting in leaf and floral organ fusions in the
dif-ferent jlo alleles. Boundary formation requires a depletion of
auxinfrom the cells encompassing the organ primordia (Heisler et
al.,2005). Boundary cells act not only as morphological barriers,
theyalso provide signals that regulate the development of
adjacenttissues (reviewed in Aida and Tasaka, 2006). The failure to
sep-arate cells with different identities, combined with a loss
ofboundary-specific gene expression, will then result in
aberrantorgan development of jlo mutants. Somewhat similar effects
werereported for the trihelix transcription factor PETAL LOSS,
which isexpressed in sepal boundaries. ptl loss-of-function mutants
de-velop fused sepals but display also a reduced organ number
aswell as polarity defects (Brewer et al., 2006).
Taken together, our data show that JLO function is requiredfor
patterning processes throughout plant development. Wedemonstrate
that JLO can act in homomeric as well as in het-eromeric complexes
with AS2 and AS1 and serves to coordinateKNOX expression and the
regulation of PIN auxin efflux carriersduring shoot and root
development in Arabidopsis.
METHODS
Plant Material and Growth Conditions
The jlo-2 (Landsberg erecta [Ler]; Bureau et al., 2010), as2-1
(CS3117, Anbackground), as2-2 (CS3118, ER background), stm-2
(N8137; Lerbackground), and bp-1 (CS30; Ler background) mutants
were obtained
from the Nottingham Arabidopsis Stock Centre. The jlo-3
(pst17018), jlo-4(pst19799), jlo-5 (pst20504), jlo-6 (pst00432),
and jlo-7 (pst13957) mu-tations are in the Nossen (No-0) background
and belong to the RIKENcollection. The origins of marker lines are
as follows: DR5rev:GFP (B.Scheres), PIN1:PIN1-GFP, PIN4:PIN4-GFP,
and PIN7:PIN7-GFP (J.Friml), AS2:GUS (J.C. Fletcher), STM:GUS (W.
Werr), and BP:GUS (M.Tsiantis). Arabidopsis thaliana plants were
grown on soil under constantlight conditions at 21°C. For root
analyses, seeds were surface sterilizedwith chlorine gas, imbibed
in 0.1% agarose, and plated onto GM medium(0.53 Murashige and Skoog
medium with Gamborg’s No. 5 vitamins[Duchefa], 0.5 g/L MES, 1%
[w/v] Suc, and 1.2% [w/v] plant agar). Plateswere incubated
vertically in a growth chamber. Nicotiana benthamianaplants were
grown for 4 weeks in a greenhouse under controlled conditions.
Binary Constructs and Plant Transformation
For protein localization and interaction studies, attB sites
were added viaPCR-mediated ligation to coding regions of JLO, AS2,
AS1, LBD31, orLBD2. PCR products were introduced into pDONR201 and
eventuallyrecombined into pABindGFP, pABindCherry, or pABindFRET
(Bleckmannet al., 2010). Binary vectors were transformed in
Agrobacterium tume-faciens GV3101 pMP90 (Koncz et al., 1984)
according to the manu-facturer’s instructions (Invitrogen). Abaxial
leaf sides of N. benthamianaplants were infiltrated as described by
Bleckmann et al. (2010). Transgeneexpression was induced 48 h after
infiltration by spraying with 20 µMb-estradiol and 0.1% Tween 20
and analyzed within 12 HAI. Production offusion proteins was
confirmed by immunoblotting (primary antibody, anti-GFP [Roche];
secondary antibody, anti-mouse ALP conjugated [Dia-nova]).
Arabidopsis plants were transformed using the floral dip
method(Clough and Bent, 1998). Transgenic plants were selected on
GMmediumcontaining hygromycin (15 mg/mL). For JLO misexpression
experiments,a LexA35S:JLO-FLAG transgene (Bureau et al., 2010) was
transformedinto Columbia-0 or as2-2 plants. Induction of transgene
expression wasperformed by spraying with 20 µM b-estradiol and 0.1%
Tween 20 andverified by immunoblot analysis (primary antibody,
anti-FLAG [Sigma-Aldrich]; secondary antibody, anti-mouse ALP
conjugated antibody [Di-anova]). For the analysis of the JLO
expression pattern, the JLO promoterregion (3273 bp upstream of the
ATG) was synthesized (Life Technolo-gies), introduced into
pDONR211, and recombined into pMDC163 (Curtisand Grossniklaus,
2003).
EFRET Measurements via Acceptor Photobleaching
N. benthamiana leaf epidermal cells and Arabidopsis root
epidermal cellswere examined with a 340 1.3–numerical aperture
Zeiss oil-immersionobjective using a Zeiss LSM 510 Meta confocal
microscope. EFRET wasmeasured via GFP fluorescence intensity
increase after photobleaching ofthe acceptor mCherry (Bleckmann et
al., 2010). The percentage change ofthe GFP intensity directly
before and after bleaching was analyzed asEFRET = (GFPafter 2
GFPbefore)/GFPafter 3 100. All photobleaching experi-ments were
performed in the nucleus. A minimum of 25 measurementswas performed
for each experiment. Significance was analyzed usinga Student’s t
test.
Yeast Interaction Studies
Full-length coding sequences or fragments of genes tested in
yeast in-teraction studies were amplified by PCR from Columbia-0
cDNA. Theforward and reverse primers used for this amplification
carried a restrictionsite that permitted cloning of the PCR product
into pGADT7, pGBKT7, orpTFT1 (Egea-Cortines et al., 1999). For
yeast two-hybrid studies, GAL4-BD and GAL4-AD clones were
transformed into the yeast strains YST1and AH109 (Clontech).
Expression of the fusion proteins was confirmed
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by immunoblotting (GAL4-BD, anti-Gal4 DNA-BD [BD Biosciences]/
anti-mouse-ALP conjugated [Dianova]; GAL4-AD, anti-HA [Roche]/
anti-rat-horseradish peroxidase conjugated [Dianova]). After
mating, interactionwas studied by plating serial dilutions of
yeasts on medium lacking Leu,Trp, His, and Ade. Three-hybrid assays
were performed in yeast strainAH109. Constructs were cotransformed
and selected on yeast syntheticdropout medium. Interactions were
assayed on quadruple dropout me-dium. All other techniques were
performed according to the Matchmakerprotocols handbook
(Clontech).
Gene Expression Analysis
Reporter gene analysis was performed in the F3 generation after
geneticcrossing. Detection of GUS activity was performed with GUS
stainingsolution (0.05MNaPO4, pH 7.0, 5mMK3[Fe(CN)6], and
10mMK4[Fe(CN)6]). Formicroscopy analysis of embryos and roots,
tissuewas clearedwith70% (w/v) chloral hydrate and 10% (v/v)
glycerol solution. For microscopyanalysis of green tissues,
chlorophyll was removed using an ethanolseries from 50% (v/v) to
100% (v/v). Tissues were then cleared with 50%to 100% (v/v) Roti
Histol, followed by overnight incubation in immersionoil. Analysis
of fluorescence reporter expression was performed usinga LSM510
Meta confocal microscope. Counterstaining of root cell wallswas
achieved by mounting roots in 10 µM propidium iodide. The
RNeasyplant mini kit (Qiagen) was used for RNA extraction. RNA was
treated withDNase (Fermentas) and transcribed into cDNA using
SuperScriptII (In-vitrogen). qRT-PCRwas performed in triplicates
using theMesa Blue SybrMix (Eurogentec) and a Chromo4 real-time PCR
machine (Bio-Rad).Expression levels were normalized to the
reference gene At4g34270(Czechowski et al., 2005).
Phenotypic Analysis and Microscopy
Analysis of embryos was performed as described by Bureau et al.
(2010).Scanning electron microscopy analysis was performed
according toKwiatkowska (2004). Root architecture was studied with
the modifiedpseudo-Schiff propidium iodide method (Truernit et al.,
2008) and imagedwith a Zeiss LSM 510 Meta laser scanning
microscope. For size meas-urements, floral organs were separated
from each other and imaged withan AxioCam ICC1 camera (Zeiss)
mounted onto a Zeiss Stemi 2000C. Tocompare cell sizes, petals were
printed with 1.5% agarose, and negativeswere examined and
photographed using an AxiocamHR camera attachedto a Zeiss Axioscope
II microscope. Images were processed in ImageJsoftware and
assembled in Adobe Photoshop.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis
GenomeInitiative or GenBank/EMBL databases under the following
accessionnumbers: AT4g00220 (JLO), AT1G65620 (AS2), AT2G37630
(AS1),AT4G00210 (LBD31), and AT1G06280 (LBD2).
Supplemental Data
The following materials are available in the online version of
this article.
Supplemental Figure 1. Molecular Analyses of the Novel jlo
Alleles.
Supplemental Figure 2. Embryo and Seedling Development in the
F1Resulting from Crosses between jlo-2 with jlo-1 and jlo-3 to
jlo-7Mutants.
Supplemental Figure 3. Floral Phenotypes of the Novel jlo
Alleles.
Supplemental Figure 4. jlo-2/+ as2-2 Double Mutant Analysis.
Supplemental Figure 5. Class I KNOX Gene Expression in
jlo-2Mutants at 15 DAG.
Supplemental Figure 6. The JLO Gain-of-Function Phenotype
Re-quires AS2 Activity.
Supplemental Figure 7. Interaction of JLO and AS2 with LBD31
andLBD2.
Supplemental Figure 8. Intracellular Localizations of LBD31
andLBD2 and Time Course Experiment.
Supplemental Table 1. Allelism Analysis.
Supplemental Table 2. Occurrence of Phenotypic Classes I to
IIIwithin the First Flowers of the jlo Alleles.
Supplemental Table 3. Suppression of the jlo-2 SAM Phenotype
bythe stm and bp Mutations.
ACKNOWLEDGMENTS
We thank Cornelia Gieseler and Carin Theres for technical
support andmembers of the R. Simon and D. Schubert laboratory for
criticalcomments. We also thank Jennifer C. Fletcher and JiHyung
Jun forproviding the AS2:GUS line, Andrea Bleckmann for the
modified pMDC7vectors and for help with confocal microscopy, Yvonne
Stahl for helpwith whole-mount RNA in situ hybridization, Marc
Somssich for helpingto clone the JLO promoter, and the Center for
Advanced Imaging atHeinrich-Heine-Universität for help with the
confocal equipment. Wethank Jiri Friml, Miltos Tsiantis, and
Wolfgang Werr for generouslysupplying plant materials. This work
was supported by a grant fromthe Deutsche Forschungsgemeinschaft
(Si 947/4-1) to R.S.
AUTHOR CONTRIBUTIONS
M.I.R. performed the experiments, and M.I.R. and R.S. designed
theresearch and wrote the article.
Received April 27, 2012; revised June 14, 2012; accepted June
27, 2012;published July 20, 2012.
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