arabidopsis jagged lateral organs acts with ...arabidopsis jagged lateral organs acts with...

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

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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.]

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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 article for color version of this figure.]

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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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