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Plant Cell. Jun 2007; 19(6): 1795–1808.
PMCID: PMC1955719

Arabidopsis JAGGED LATERAL ORGANS Is Expressed in Boundaries and Coordinates KNOX and PIN Activity[W]

Abstract

Plant lateral organs are initiated as small protrusions on the flanks of shoot apical meristems. Organ primordia are separated from the remainder of the meristem by distinct cell types that create a morphological boundary. The Arabidopsis thaliana gain-of-function mutant jagged lateral organs-D (jlo-D) develops strongly lobed leaves, indicative of KNOX gene misexpression, and the shoot apical meristem arrests organ initiation prematurely, terminating in a pin-like structure. The JLO gene, a member of the LATERAL ORGAN BOUNDARY DOMAIN gene family, is expressed in boundaries between meristems and organ primordia and during embryogenesis. Inducible JLO misexpression activates expression of the KNOX genes SHOOT MERISTEMLESS and KNAT1 in leaves and downregulates the expression of PIN auxin export facilitators. Consequently, bulk auxin transport through the inflorescence stem is drastically reduced. During embryogenesis, JLO is required for the initiation of cotyledons and development beyond the globular stage. Converting JLO into a transcriptional repressor causes organ fusions, showing that during postembryonic development, JLO function is required to maintain the integrity of boundaries between cell groups with indeterminate or determinate fates.

INTRODUCTION

Lateral organs of a higher plant are initiated from small cell groups on the flanks of the dome-shaped shoot apical meristem (SAM). The SAM acts as the stem cell niche that provides new cells to support continuous growth and organ development. Maintenance of an active shoot meristem requires expression of homeobox KNOX genes, such as SHOOT MERISTEMLESS (STM) of Arabidopsis thaliana, which are excluded from organ primordia (Long et al., 1996). Loss-of-function mutations in STM result in embryos lacking a meristem due to misexpression of the MYB transcription factor ASYMMETRIC LEAVES1 (AS1) (Byrne et al., 2000). AS1 is expressed in organ initials and physically interacts with AS2, a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family in Arabidopsis, to inhibit KNOX gene expression, thus guiding primordia toward differentiation (Ori et al., 2000; Byrne et al., 2002). Recessive mutations in as1 or as2 allow for ectopic KNOX gene expression in leaves, resulting in the formation of serrate or strongly lobed leaves, while ectopic expression of AS2 (and other LBD genes) represses KNOX gene expression (Lin et al., 2003; Chalfun-Junior et al., 2005). How these contrasting patterns of gene expression are established and maintained and how boundaries between immediately adjacent gene expression domains are set are only poorly understood.

Morphological boundaries are established at an early stage of organ primordium formation that separate the organ from the remainder of the meristem but are also the sites for initiation of axillary meristems later in development (Aida and Tasaka, 2006). Cells in the boundary are distinctly narrow and elongated and divide only infrequently. Genes expressed in the boundary may regulate both meristem and organ development. The LATERAL ORGAN BOUNDARY (LOB) gene, the founding member of the LBD gene family, encodes a putative transcription factor that is expressed in boundaries of the shoot but also at the base of secondary roots (Shuai et al., 2002). LOB is positively regulated by the meristem-specific KNOX gene KNAT1 and by the organ-specific gene AS2 (Lin et al., 2003). However, LOB loss-of-function mutants are aphenotypic, and the precise role of LOB in boundary or meristem development remains to be shown.

Similar to LOB, CUP-SHAPED COTYLEDON (CUC1), CUC2, and CUC3 are also expressed in boundaries. They encode related NAC domain transcription factors that promote morphological separation of lateral organs through growth repression (Aida et al., 1997; Vroemen et al., 2003). CUC genes act in part redundantly; double mutant combinations (e.g., cuc1 cuc2) produce fused cotyledons and no embryonic SAM because CUC activity is already required during early embryogenesis to activate and delineate expression of STM, which is required for SAM establishment (Aida et al., 1999; Takada et al., 2001).

The position of an organ appears to be regulated by the distribution of the phytohormone auxin, such that local auxin accumulation permits organ primordia initiation (Reinhardt et al., 2000, 2003), and controlled redistribution of auxin is achieved through directional auxin transport. Proteins of the PIN family are membrane-localized auxin efflux carriers that are themselves subject to feedback regulation by auxin (Paciorek et al., 2005). pin mutant shoots fail to accumulate auxin locally and do not initiate lateral organs. However, creating artificial auxin peaks by local auxin application on pin mutant meristems can induce organ formation (Reinhardt et al., 2003), showing the importance of auxin distribution and signaling for patterning and organogenesis at the shoot tip. Furthermore, regulation of auxin transport and gene expression at boundaries are interdependent, since PIN1 gene expression is downregulated at boundaries, and mutants that affect auxin distribution, such as pin1, misexpress the boundary gene CUC1 (Vernoux et al., 2000; Furutani et al., 2004). However, how auxin transport and signaling are coordinated with the required downregulation of meristem-specific genes during lateral organ formation is not yet understood.

Here, we show that the LBD gene JAGGED LATERAL ORGANS (JLO) is first required for progression of embryogenesis beyond the globular stage. At later stages, JLO is expressed in boundaries, and misexpression experiments reveal that JLO activates KNOX gene expression but represses PIN1. We propose that JLO acts from the boundary to orchestrate the drastic changes in gene expression that entail the initiation of plant lateral organs.

RESULTS

Isolation and Phenotype of the jlo-D Mutant

The jlo-D mutant was identified in an activation tagging mutagenesis program. Briefly, a T-DNA carrying a modified Spm transposable element (dSpm-Act) with four copies of the cauliflower mosaic virus 35S promoter (CaMV35S) enhancer element flanked by a selectable marker gene conferring resistance to the herbicide BASTA was integrated into the Arabidopsis genome via Agrobacterium tumefaciens–mediated transformation (Kirch et al., 2003). dSpm-Act excision and reinsertion at novel positions in the genome resulted in activation of gene expression in the immediate neighborhood. The jlo-D mutant (Figure 1A) was identified in this transposon mutagenesis due to its small size and aberrant leaf shape (Figure 1B, top leaf). Development of jlo-D mutant seedlings starts to diverge from the wild type ~10 to 20 d after germination (DAG). At this stage, soil-grown plants have developed 6 to 10 visible leaves. Consecutively formed leaves on jlo-D mutants grow progressively shorter than wild-type leaves (wild-type leaf length, 3.5 ± 1.0 cm [sd], n = 46; jlo-D, 2.4 ± 0.7 cm [sd], n = 43). In addition, the blade-to-petiole ratio is increased in jlo-D mutants (wild type, 1.6; jlo-D, 3.7) (Figure 1B). Furthermore, jlo-D leaves remain smaller and lobed. Stem elongation of the inflorescence is reduced, giving the plant a bushy phenotype. The first 10 to 17 flowers develop normally. In later flowers, the pistil formed from the fusion of two carpels grows unevenly and bends due to the lack of a valve on one side (Figures 1C and 1D). Occasionally, flowers lacked one or more sepals or stamens (Figure 1E), or organs failed to develop to maturity, resulting in sterile flowers and reduced overall fertility.

Figure 1.
Phenotype of jlo-D Mutants.

On average, the shoot meristem of jlo-D mutants arrests activity after formation of ~13 flowers (12.7 ± 2.2, n = 10), terminating in a pin-like structure that lacks organ primordia (Figure 1F). The last organ to be produced before meristem arrest often appears radially symmetrical (Figure 1G, arrowhead). The jlo-D mutant is not fully penetrant, with only 60 to 70% of all plants showing a mutant phenotype.

Functional Analysis of jlo-D Mutants

To characterize the developmental defects of jlo-D mutant plants, we first analyzed the expression of genes directing meristem functions. The termination of shoot meristem activity in jlo-D could be caused by a failure to maintain an active stem cell population. However, RNA in situ hybridizations revealed strong expression of the stem cell marker CLV3 at the shoot tip (Figure 1H). Surprisingly, stem cell number appears increased in jlo-D mutants compared with the wild type, suggesting a delayed exit of cells from the central to the peripheral zone. To analyze if the feedback regulatory circuitry between WUS and CLV3 was affected in jlo-D mutants, we analyzed double mutants of jlo-D with clv3-2 and wus. clv3 mutants accumulate stem cells in shoot and floral meristems, resulting in stem fasciation, increased production of floral organs, and partial indeterminacy of floral meristems (Clark et al., 1995). clv3-2 jlo-D double mutant plants formed lobed rosette leaves, flowers with an increased number of floral organs, and a larger inflorescence meristem that terminated in an expanded, pin-like structure with arrested organ primordia, suggesting additive effects (Figure 1I). Furthermore, jlo-D wus plants displayed the wus phenotype, indicating that WUS is functional in a jlo-D background and that the developmental arrest of jlo-D meristems is not caused by increased signaling via the CLV pathway and downregulation of WUS (Brand et al., 2000).

In wild-type plants, STM is expressed in meristematic tissues (Figure 2A) and in the medial ridge of the gynoecium but downregulated at sites of organ formation (Long et al., 1996; Skinner et al., 2004). In jlo-D mutants, STM was strongly expressed throughout the arrested SAM (Figure 2B). We also noted increased and expanded STM expression in the medial ridge and in developing ovules (Figures 2C and 2D). Interestingly, STM was kept strongly upregulated in the medial ridge of univalved pistils in jlo-D mutants, thus possibly impairing initiation of the second carpel (Figure 2E).

Figure 2.
Ectopic JLO Expression Induces STM Expression.

Molecular Analysis of the jlo-D Mutant

Genomic DNA flanking the dSpm-Act transposable element insertion that is responsible for the jlo-D mutation was isolated by inverse PCR and sequenced. The dSpm-Act element is located between At4g00210 and At4g00220, two immediately adjacent genes that are transcribed in opposite orientations (see Supplemental Figure 1 online). On RNA gel blots with total RNA and by RT-PCR, we detected high levels of At4g00220 transcripts in jlo-D mutant leaves but no expression in wild-type leaves (data not shown), whereas At4g00210 RNA was not expressed in leaves of mutant or wild-type seedlings. Furthermore, misexpression of At4g00210 from the constitutive CaMV35S promoter in transgenic plants did not visibly affect plant development. By RNA in situ hybridizations on jlo-D tissue sections, we detected transcripts of At4g00220 throughout the plant (Figure 3P). Since expression of At4g00210 was unaffected by the dSpm-Act insertion, we concluded that the jlo-D phenotype was caused by ectopic expression or upregulation of At4g00220. Interestingly, At4g00210 and At4g00220 encode closely related proteins (LBD31 and LBD30, respectively) that belong to the LBD family of putative transcription factors (Shuai et al., 2002). The founding member of this family, LOB, was shown to be nuclear localized and expressed in the boundary regions that separate young organ primordia from meristems. Using transient gene expression, we found that fusions of the JLO protein with green fluorescent protein (GFP) were localized to the nucleus (data not shown).

Figure 3.
JLO Is Expressed in Boundaries.

JLO Expression Pattern

We analyzed the expression pattern of JLO during wild-type development by RNA in situ hybridization. JLO transcripts are found at the sites of organ initiation at the peripheral zone of the SAM. On longitudinal sections through the inflorescence apex, JLO is expressed in a roughly triangular domain of four to six cells wide and three to four cells deep. The expression domain encompasses the adaxial half of the young stage 1 flower primordia (Figures 3A to 3H). Cross sections reveal that JLO RNA marks the tip of flower primordia when they are initiated and in deeper sections marking the boundary between initiating flower primordia and the meristem (Figures 3I to 3M). However, JLO RNA is not detectable in the boundary between late stage 2 floral meristems and the inflorescence meristem. In flowers, JLO RNA is first detected when sepal primordia are initiated, and at stage 3, when sepals are clearly separated from the floral meristem, JLO is expressed in a narrow band approximately two cells wide and three cells deep that marks the boundary between sepals and meristem. We did not detect expression later than stage 4 in the meristem-to-organ boundaries. During carpel development, JLO was weakly expressed in ovules (Figures 3N and 3O). We also investigated the publicly available expression analysis data obtained with Affymetrix microarrays. In the developmental data set of AtGenExpress, JLO was found to be significantly expressed during seed development. We were unable to detect JLO RNA by in situ hybridizations on sectioned seeds, indicating that expression is too low for our detection system. However, using RT-PCR, we found JLO to be expressed in embryos (data not shown).

Overexpression of JLO Phenocopies the jlo-D Mutant Phenotype

To confirm that the jlo-D phenotype is caused by misexpression of JLO, we expressed a JLO cDNA clone from the CaMV35S promoter (35S:JLO). Surprisingly, we were able to generate only six transgenic plants from several transformation experiments, suggesting that a high level of JLO expression may strongly interfere with normal development. Surviving 35S:JLO plants developed small and strongly lobed leaves, a short inflorescence and curved pistils. In addition, sepal and stamen failed to mature, and plants were therefore sterile (Figures 2F and 2G). The floral phenotypes of JLO misexpressors strongly resembled plants misexpressing STM after floral induction (Figure 2H). Together, 35S:JLO plants showed a similar, albeit somewhat stronger phenotype than jlo-D mutants, which could be due to higher expression of JLO in the 35S:JLO plants. Misexpression of other members of the LBD gene family has previously been shown to affect axial patterning of lateral organs and cell fate. However, scanning electron microscopy revealed that cell size and shape on the adaxial and abaxial surfaces were unaffected by JLO misexpression in both jlo-D and 35S:JLO plants (data not shown).

To circumvent deleterious effects of JLO misexpression, we expressed JLO as a translational fusion with the hormone binding domain of the glucocorticoid receptor (35S:JLO-GR). Four transgenic lines were identified that responded to induction with dexamethasone (Dex). Single inductions were sufficient to phenocopy 35S:JLO plants, including shoot and lateral meristem termination (Figure 2I). Repetitive inductions with Dex caused premature arrest of organ development, and ~10% of the seedlings bleached out and wilted, indicating that gross misexpression interferes with cellular processes essential for survival (data not shown). We also noted that epidermal trichomes were strongly misshapen (see below).

JLO Is Required for Embryo Development

To investigate the role of JLO during normal development, we analyzed a mutant line that carries a T-DNA insertion in the JLO gene (SALK_020930, allele named jlo-1; see Supplemental Figure 1 online). Plants obtained from the Arabidopsis stock center were shown to be heterozygous for the jlo-1 allele. However, genotyping by PCR revealed no homozygous plants in the progeny after selfing of jlo-1/+ plants, indicating that JLO function may be required for development to the seedling stage. Indeed, analysis of siliques from jlo-1/+ plants revealed that approximately one-quarter of all embryos did not develop to maturity (647 wild type:194 mutant; wild type:mutant = 3.3:1; deviation from the 3:1 ratio is not significant with a χ2 value = 1.675; 0.5 < P < 0.10) (Figure 4A). The close to 3:1 segregation ratio indicates that homozygous jlo-1/jlo-1 individuals die as embryos, suggesting that JLO expression is essential for normal embryogenesis.

Figure 4.
JLO Is Required for Embryonic Development.

To further analyze the time point of developmental arrest, we collected siliques from jlo-1/+ plants and investigated the development of the embryos. In each silique, ~25% of all embryos (jlo-1/jlo-1) were delayed or arrested compared with their siblings. First deviation from wild-type development was observed at the octant stage, when mutant proembryos consisted of only three or four cells (Figures 4B and 4H). When wild-type embryos had reached early heart stage with clearly distinct cotyledon primordia and an embryonic root pole, mutants consisted of a ball-shaped embryo without a clear organization and lacking elongated vascular initials that are observed in the wild type by this stage (Figures 4E and 4K). Later cell divisions appeared irregular, and although the cells kept proliferating, overall growth was delayed. The suspensor of mutants occasionally consisted of several cell rows, indicating that cell division patterns were disturbed but not generally inhibited. When wild-type embryos had reached the bent cotyledon stage, jlo-1 mutant embryos were small and round and had apparently arrested development (Figures 4G and 4M).

We analyzed a second insertion line that carries a Ds transposable element insertion in the 3′ part of the first exon (JIC_GT.9713, allele named jlo-2). After selfing of jlo-2/+ heterozygous plants, no homozygous jlo-2/jlo-2 mutants could be identified by genotyping. Analysis of developing siliques revealed ~17% missing seeds in the jlo-2/+ mutant compared with the wild type (see Supplemental Figure 2 online). Among the developing seeds, 12% arrested at the globular stage, similar to jlo-1 mutant embryos.

The severe defects of jlo-1 and jlo-2 embryos indicated that JLO is already required during early stages of embryonic pattern formation.

Overexpression of a Dominant-Negative Version of JLO (JLO-DN)

The embryo lethality of homozygote jlo-1 and jlo-2 mutants did not permit us to uncover the functions of JLO at later stages of development. We therefore created a dominant-negative version of JLO (CaMV35S:JLO-DN) to inactivate JLO function. We expressed a fusion protein between JLO and the EAR domain, a short motif found in several transcriptional repressors, in transgenic plants (Hiratsu et al., 2003). We argued that if JLO acts as a transcriptional regulator, conversion into a transcriptional repressor would result in the repression of its target genes and therefore phenocopy a hypomorphic or amorphic phenotype.

The majority of transgenic plants (n = 60) obtained appeared wild-type. However, ~20% of the seedlings showed a partial fusion of the cotyledons (Figures 5B and 5C). Leaves that were formed at later stages curled upwards, had thick blade margins, and did not expand properly (Figures 5D and 5E). Some seedlings initiated only filamentous organs that failed to grow along the proximo-distal axis (Figure 5F). We analyzed the epidermal surface structure of leaves by scanning electron microscopy. The adaxial epidermal cells of young wild-type leaves are variable in size and shape and interspersed with stomata and stomatal meristemoids. Abaxial cells are more elongated and strongly interdigitized. Cells on the adaxial surface of JLO-DN leaves resembled adaxial cells of the wild type, but abaxial cells remained compact and small, indicating that JLO-DN mutants have failed to establish abaxial identity (Figures 5G to 5J). Organs initiated at later stages failed to grow out, and the plants died eventually before flowering.

Figure 5.
A Dominant-Negative Version of JLO Inhibits Embryo and Boundary Development.

There are two possible explanations for the apparent differences between the phenotypes of JLO-DN and jlo-1 mutants. First, the JLO-DN mutants could be leaky (i.e., they could allow the expression of some JLO target genes). Second, transgenic plants expressing JLO-DN at high levels could be embryo lethal and will therefore not be discovered when selecting for transgenic plants at the seedling stage. We then expressed JLO-DN from an ethanol-inducible promoter (35S:iJLO-DN) and induced transgene activity using ethanol vapor. While transgene activity was apparently too low to affect most stages of development, we noted that seed set was severely reduced due to abortion, suggesting that expression of JLO-DN can block embryo development (Figure 5A).

Target Genes Regulated by JLO

Using ATH1 Affymetrix microarrays, we identified candidate target genes for regulation by JLO. Probes were generated from leaf RNAs that were harvested at 0 or 25 h after induction of JLO-GR activity. We found at least twofold changes in expression levels for 477 genes. We selected several genes with assigned functions for further expression analysis and validation by quantitative RT-PCR (qRT-PCR) (Table 1). It is noteworthy that two other genes of the LBD family whose functions are unknown, LBD41 and LBD42, were strongly upregulated (Shuai et al., 2002).

Table 1.
Genes Regulated by JLO

Cell Cycle Regulators

We found that two cyclin genes, CYCD3;1 and CYCD3;2, were upregulated (Table 1). CYCD3;1 misexpression only in trichomes was shown to induce DNA replication but also further cell divisions (Schnittger et al., 2002). However, misexpression in leaves showed that CYCD3;1 inhibits endocycles and cellular differentiation (Dewitte et al., 2003). The role of CYCD3;2 has not been analyzed in detail. We noted that inducible JLO misexpression drastically affected trichome development. Approximately 48 h after Dex treatment, trichomes with massively enlarged bases became visible (Figure 6A, left panel). Trichomes are large and terminally differentiated cells that have undergone several rounds of endomitosis. Comparison of nuclei sizes and DNA content in isolated trichome cells of wild-type and induced JLO-GR plants showed an increase in DNA content and nucleus size in the enlarged trichomes (nucleus size of control trichomes, 169 μm2; nucleus size of JLO-GR trichomes, 278 μm2; n = 40). Wild-type trichome ploidy levels average 32C, but JLO-GR trichome nuclei underwent two additional rounds of endoreduplication to reach an average of 128C (Figure 6A, right panel). Thus, induced overexpression of JLO transiently interferes with cell cycle regulation in trichomes. However, analysis of nuclear DNA content by flow cytometry revealed a different response of leaf cells to increased JLO expression. We compared ploidy levels in leaf 6 (L6) to L17 at 42 DAG. In the wild type, ploidy levels increase steadily with leaf age due to endoreduplication, but this increase was reduced or delayed when JLO was misexpressed (Figure 6B), indicating that JLO activity interferes with cell cycle regulation and/or delays differentiation.

Figure 6.
JLO Misexpression Affects Endomitosis.

AS1 and KNOX Genes

The lobed leaf phenotype of jlo-D mutants indicated that KNOX genes, such as STM and KNAT1, are possible targets for activation by JLO. Consistent with this, we observed enhanced expression of STM in several tissues of jlo-D mutants (Figure 2). AS1 is required to restrict KNAT1 gene expression to the meristem, and as1 mutants grow lobed leaves that misexpress KNAT1 and, albeit to a lesser extent, STM (Semiarti et al., 2001). To analyze for possible interactions between jlo-D and AS1, we first constructed jlo-D/as1 double mutants. We could not easily distinguish jlo-D/as1 leaves from the as1 or jlo-D single mutant leaves, suggesting either interference with the same genetic pathway or that jlo-D acts by repressing AS1. To identify AS1-independent activities of JLO, we introduced the inducible JLO-GR transgene into as1 mutants. Uninduced as1 and as1/JLO-GR seedlings were indistinguishable (Figures 7A and 7C). Activating JLO by spraying seedlings with 1 μM Dex from 14 DAG onwards (Figure 7D) caused a strong enhancement of the as1 leaf phenotype. The petioles remained short, and seedlings formed a very dense leaf rosette. Furthermore, we noted that all induced JLO-GR plants, independent of AS1 activity, carried aberrantly shaped trichomes (Figure 7D, inset). Thus, it is unlikely that JLO acts solely by repressing AS1 in leaves to allow ectopic KNOX gene activity.

Figure 7.
JLO Acts Independently of AS1.

KNOX and PIN1 Expression

In wild-type plants, PIN1 RNA is found in organ primordia, young floral meristems, and vascular tissue (Vernoux et al., 2000). Furthermore, PIN1 expression is promoted by auxin (Paciorek et al., 2005). Interestingly, several reports illustrated that misexpression of KNOX genes impedes polar auxin transport, suggesting that auxin transport or signaling and KNOX genes affect each other (Tsiantis et al., 1999; Scanlon et al., 2002; Scanlon, 2003; Hay et al., 2004).

To investigate regulation of KNOX and PIN genes through JLO, we used qRT-PCR to monitor the changes in expression levels of STM, KNAT1, and PIN1 after inducible misexpression of JLO using the 35S:JLO-GR transgene. RNA was extracted from inflorescences at 0, 3, 9, 12, and 25 h after induction (HAI). qRT-PCR revealed a twofold increase of STM expression in inflorescences within 25 HAI. In leaves, RNA levels of PIN1 declined rapidly within 3 HAI, with only residual expression until 25 HAI. Both KNAT1 and STM are expressed at only very low levels in leaves before induction, but their RNA levels increased 10- to 20-fold, respectively, within 25 HAI, reaching 40% PIN1 preinduction levels (Figure 8). The reduction of PIN1 RNA levels within 3 HAI preceded the induction of STM and KNAT1; however, we have so far analyzed only steady state RNA levels and can therefore not deduce a temporal order of activation and repression.

Figure 8.
JLO Controls KNOX and PIN Expression.

Auxin Transport and Signaling

In addition to PIN1, PIN3, PIN4, and PIN7 expression was also downregulated between 8- and 18-fold after induction of JLO expression, while PIN2, PIN5, PIN6, and PIN8 were not affected (Table 1). Such a drastic transcriptional repression of PIN efflux facilitators could help to explain the production of naked inflorescence meristems in jlo-D mutants. Organs are initiated in the peripheral zone at sites where auxin accumulates. Without this buildup of auxin peaks, organ production is inhibited. Application of auxin to the almost naked (without organs) SAM of pin1 mutants can rescue organ formation (Reinhardt et al., 2000). However, auxin applications failed to induce organ formation on the arrested SAM of jlo-D mutants (data not shown), indicating that jlo-D mutants are compromised in auxin transport, sensing or signal transduction. The phenotypic resemblance between pin1 and jlo-D mutant shoot meristems prompted us to analyze jlo-D pin1 double mutants (Figures 9A and 9B). Rosette leaves of pin1 mutants show multiple vascular strands in the center, and leaves appear broader than in the wild type. The jlo-D mutation drastically enhanced this phenotype: jlo-D pin1 double mutants produced only one large, strongly lobed leaf and occasionally a reduced, filamentous organ. Shoot development arrested without the formation of an inflorescence meristem (Figures 9A and 9B). Thus, the jlo-D mutant strongly enhanced the pin1 mutant phenotype, indicating that auxin transport may be further reduced in the double mutants.

Figure 9.
JLO Restricts Auxin Transport.

In wild-type plants, basipetal auxin transport along the inflorescence axis depends on PIN1. If JLO represses PIN gene expression, we should expect a reduction in overall auxin transport activity. We measured auxin transport activity in Columbia and JLO-GR plants using radiolabeled indole-3-acetic acid (IAA). For each assay, 10 inflorescence segments from either Dex-treated wild-type or JLO-GR plants were incubated with 3H-IAA, and the amount of IAA transported from the apical to the basal end of the stem segment within 18 h was quantified. We found that bulk auxin transport was reduced in JLO-GR plants down to 30% of wild-type levels.

We then analyzed the activity of the auxin-sensitive DR5rev:GFP reporter to increased JLO expression (Figures 9D to 9K). The JLO-GR transgene was introduced into DR5rev:GFP transgenic plants, and GFP fluorescence was studied by confocal microscopy. Cross sections through stem tissue revealed strongly reduced GFP signal compared with the wild type (Figures 9D and 9H), which could be explained by the strong reduction in apical-basal auxin transport through the stem. During leaf development, DR5rev:GFP expression is first seen at the leaf tips, the hydathodes, leaf marginal tissue, and vascular tissue (Scarpella et al., 2006) (Figures 9E and 9F). Consistent with a reduction of auxin transport through the vasculature, DR5rev:GFP is increased at synthesis sites (hydathodes) but strongly reduced in the vasculature tissue of JLO-GR leaves. Auxin accumulation is still detected at the tips of floral organs in a wild-type pattern but at lower levels (Figures 9F and 9J). Cell division patterns of JLO-GR root meristems are less regular than in the wild type, and the layered organization of the columella is disturbed. In wild-type roots, DR5rev:GFP is strongly expressed around the quiescent center and the proximal columella layers (Figures 9G and 9K). The quiescent center expression of DR5rev:GFP is maintained in JLO-GR roots, but expression in the columella is slightly increased and shifted to a more distal position. Auxin is transported to and concentrated at the root meristem via the activities of PIN1, PIN2, PIN3, PIN4, and PIN7 (Vieten et al., 2005). Increased DR5 activity in the columella could reflect reduced PIN4 and PIN7 expression in this region. However, there is redundancy of PIN protein functions, and most pin mutants are aphenotypic in the root, with the exception of pin2 (Muller et al., 1998). Ectopic expression and functional complementation of PIN proteins will therefore dampen the effects of a partial PIN gene repression by JLO, allowing sustenance of an almost normal auxin distribution (Vieten et al., 2005).

JLO activity is required for progression of embryo development beyond the globular stage. In wild-type embryos of this stage, DR5rev:GFP activity is detected in the upper suspensor and the hypophysis that gives rise to the root meristem (Figure 4N). However, 60% of the jlo-1 mutant embryos (Figure 4O; see Supplemental Table 1 online) showed activation of the DR5rev promoter only in the suspensor but not in the hypophysis, indicating that JLO is required for auxin dependent patterning of the early embryo.

DISCUSSION

The founding member of the LBD gene family, LOB, was identified as an enhancer trap line that is expressed in the boundaries between meristems and organs but also at the base of young lateral roots. LOB was demonstrated to be a nuclear protein, and LBD proteins were suggested to be involved in transcriptional regulation.

Whether LBD proteins activate or repress transcription is so far unclear: Assays in a yeast system showed that the LOB domain of the rice (Oryza sativa) ARL1 protein represses transcription, while the C-terminal domain activates it (Liu et al., 2005). AS2 was shown to interact with the MYB protein AS1, which associates with the histone chaperone HIRA to silence KNOX genes in lateral organs by modulating chromatin structure (Phelps-Durr et al., 2005). Reduced expression of KNOX genes was observed after misexpression of AS2 (LBD6) or ASL1 (LBD36) (Semiarti et al., 2001; Lin et al., 2003; Chalfun-Junior et al., 2005). Consistent with a role in regulation of gene expression, we observed JLO-GFP fusion proteins in the nucleus. Furthermore, the C-terminal domain of JLO acts as a strong transcriptional activator in yeast cells (data not shown).

We isolated JLO as an important developmental regulator by its gain-of-function phenotype. The jlo-D mutant that we identified by activation tagging was first characterized by its lobed leaves and early meristem arrest. Lobed leaves are often formed when meristem-specific KNOX genes are ectopically expressed in the leaf domain, suggesting a role for JLO, as for the related LBD gene AS2, in KNOX gene regulation. However, AS2 and JLO must act antagonistically because AS2 represses KNOX expression, whereas JLO misexpression induces ectopic STM and KNAT1 expression. In wild-type shoot and floral meristems, STM expression is downregulated at sites of organ formation. Extended STM expression in both jlo-D and JLO-GR plants first interferes with the specification of organ founder cells at lateral positions, causing the formation of univalved pistils. Apparently, STM misexpression is also responsible for organ developmental defects at later stages because similar defects were seen in STM-GR plants.

Development of most jlo-D plants arrested with a naked pin-shaped meristem that lacked any lateral organ primordia. We did not observe such meristem phenotypes in STM-overexpressing plants, suggesting that JLO expression must regulate other target genes in addition to KNOX genes. Using an inducible JLO misexpression line to assay for changes in gene expression, we found that genes of the PIN family were strongly repressed after JLO induction. Mutations in the PIN1 gene cause fusions of vasculature during leaf development and an arrest of lateral organ initiation from the shoot meristem (Okada et al., 1991; Mattsson et al., 1999). Reduced expression of PIN1 could account for the naked meristem phenotype of JLO-overexpressing plants. However, jlo-D enhanced the vascular phenotypes of pin1 in double mutant combinations. pin1 mutants were reported to maintain between 7 and 14% of wild-type auxin transport capacity in the stem axis due to either residual PIN1 expression or activity of redundantly acting members of the PIN protein family (Okada et al., 1991; Vieten et al., 2005). Enhancement of pin1 phenotypes could be explained by negative regulation of other PIN genes by JLO, which we confirmed for PIN3, PIN4, and PIN7. Consistent with diminished auxin export capacity, bulk auxin transport through the stem axis was reduced to ~30% of wild-type levels after JLO misexpression. If auxin transport to and within the shoot meristem is similarly reduced, auxin should fail to accumulate at sites of future organ primordia, and no further organs are initiated. However, we also noted that in contrast with pin1 mutant meristems (Reinhardt et al., 2000), external application of auxin to naked jlo-D meristems did not compensate for such local auxin deficiencies, indicating that increased JLO expression could also interfere with auxin perception or signal transduction. Importantly, PIN expression itself responds to auxin (Vieten et al., 2005), and we cannot easily distinguish between a direct repression of PIN gene expression by JLO or interference of JLO with auxin signal transduction, causing a reduced expression of some of the PIN genes by an indirect mechanism. As evidenced by the DR5rev:GFP pattern, auxin signaling is not generally inhibited by JLO expression, whereas export of auxin from synthesis sites was impeded. These results indicate that JLO regulates PIN gene activity during plant development; however, whether JLO acts directly or indirectly upon PIN gene expression requires further experimentation.

JLO is already needed at earlier stages of development for embryo organization because jlo mutant embryos showed aberrant cell division patterns and failed to develop beyond the globular stage. Embryo patterning is controlled by the localized redistribution of auxin (Weijers et al., 2005), and inactivation of the auxin efflux carrier system in quadruple pin1 pin3 pin4 pin7 mutants causes aberrant cell divisions, patterning defects, and embryo lethality at the late globular stage, phenotypically resembling jlo mutant embryos (Friml et al., 2003; Blilou et al., 2005).

Specification of the hypophysis, the initial step in root meristem generation, is controlled by MONOPTEROS (MP) and BODENLOS (BDL) (Weijers et al., 2006). MP and BDL act antagonistically and promote auxin transport from the proembryo to the adjacent hypophysal cell. This signaling process is disturbed in jlo mutants, suggesting that JLO acts downstream of the auxin response factor MP during embryogenesis.

Our data showed that JLO misexpression during shoot development has opposite effects upon KNOX and PIN gene expression. However, can JLO regulate these genes also during wild-type development? In the shoot, JLO is expressed in the adaxial half of young organ primordia and later only in the boundary between initiating organs and the meristem. Interestingly, a detailed expression analysis by live imaging of Arabidopsis meristems showed that STM is downregulated at locations were PIN1 is upregulated (Heisler et al., 2005). STM is expressed in the meristem but not in primordia, whereas PIN1 expression is low in the meristem and marks early on sites of organ initiation. Once primordial position is determined, STM is specifically upregulated in the boundaries, while PIN1 is excluded from the boundary domain (Heisler et al., 2005). This is entirely consistent with our observation of JLO expression in the boundary and its antagonistic effects on STM and PIN expression, indicating that JLO plays a prominent role in generating a boundary-specific pattern of KNOX and PIN gene expression.

JLO promoted endocycles in trichomes, while delaying or inhibiting them in leaf cells. These opposite effects could be explained if the regulation of endocycles differs between these two cell types. Indeed, misexpression of CYCD3;1 was shown to induce DNA replication and cell divisions in trichomes (Schnittger et al., 2002) but to inhibit endoreduplication and differentiation in leaf cells (Dewitte et al., 2003). Previous studies had detected CYCD3;1 RNA early at the adaxial side of primordia but later only faintly in boundaries. This partial overlap with JLO expression suggests that regulation of CYCD3;1 by JLO depends on the developmental context (Breuil-Broyer et al., 2004).

Expression of JLO-DN in transgenic plants caused seedling lethality and various degrees of organ fusion during early development, confirming that JLO is required to repress growth between developing organs. The fusion of cotyledons that we found in some JLO-DN plants is reminiscent of cuc1 cuc2 double mutant seedlings (Aida et al., 1997; Vroemen et al., 2003). Like JLO, CUC1 and CUC2 are expressed first during early embryogenesis and later confined to regions between organs where they are required for organ separation. JLO and CUC genes share another role in the activation of KNOX gene expression (Takada and Tasaka, 2002).

Organ initiation requires a radical change in gene expression profiles and the establishment of morphological boundaries that separate cell groups with determinate (organ) from those with an indeterminate fate (meristem). The sites of leaf initiation are determined by two apparently distinct processes: auxin accumulation and loss of meristem-specific gene expression in organ anlagen. We showed here that JLO, encoding an LBD transcription factor, can coordinate these two processes by controlling both auxin transport and the expression of KNOX genes.

METHODS

Growth Conditions

Arabidopsis thaliana plants were grown on soil or 0.5× Murashige and Skoog agar supplemented with 1% sucrose under either a 10-h-light/14-h-dark regime (short-day conditions) or a 16-h-light/8-h-dark regime (long-day conditions) at 22°C.

Genetics

Details of the TAMARA transposable element activation tagging system are available upon request (Schneider et al., 2005). The jlo-D mutant line identified in the TAMARA population carried a single dSpm/En transposon insertion in the Columbia background. For the generation of double mutant lines, the transmission of the jlo-D allele in various mutant backgrounds was followed by use of the BASTA resistance marker in the dSpm-Act element. All phenotypes were analyzed among BASTA-resistant F3 progeny. The same procedure was followed to introduce reporter genes into jlo-D mutants.

Molecular Techniques

The genomic insertion site of the dSpm-Act element in jlo-D mutant plants was determined as described (Kirch et al., 2003). Conditions for qPCR and RT-PCR were described previously (Muller et al., 2006). For microarrays, RNA was isolated from seedling leaves 14 DAG using TRIzol reagent (Invitrogen). RNA quality was assayed using a Bioanalyzer 2100 (Agilent). Affymetrix Ath1 microarrays were hybridized according to the manufacturer's instructions, and expression estimates were calculated using RMAExpress for background adjustment and quantile normalization. Genes up- or downregulated at least twofold were further analyzed. Data files are available through the Nottingham Arabidopsis Stock Centre (NASCARRAYS-422). For annotation and gene identification, The Arabidopsis Information Resource (http://www.arabidopsis.org/), the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.org/), and MATDB databases were accessed (http://mips.gsf.de/proj/thal/db/index.html).

Semi-automated nonradioactive in situ hybridization experiments were performed using an In Situ Pro VS robot (Intavis) following protocols suggested by the manufacturer. Sequence analyses were performed using the GCG Package (version 4.0; Genetics Computer Group) and VECTOR NTI suite (Invitrogen).

Chimeric Constructs and Plant Transformation

The JLO (At4g00220) and LBD31 (At4g00210) coding sequences were amplified from cDNA by PCR and cloned into pMDC32 for overexpression analysis and pMDC44 for localization of the GFP fusion proteins (http://www.unizh.ch/botinst/Devo_website/curtisvector/). For CaMV35S:JLO-GR, the JLO open reading frame was inserted as a BamHI-SpeI fragment into the binary vector pBI-dGR in frame with the hormone binding domain of the glucocorticoid receptor. The JLO-EAR fusion was generated using appropriate primer sequences. All details of vector construction and primer sequences used are available upon request. Transgenic plants were generated by vacuum infiltration of Arabidopsis ecotype Columbia recipient plants using Agrobacterium tumefaciens strain GV3101 (Bechtold and Pelletier, 1998), and plants were selected using resistance against the herbicides kanamycin, hygromycin, or BASTA.

Histological, Scanning Electron Microscopy, and GUS Analysis

Detection of β-glucuronidase (GUS) activity, tissue preparations, and RNA in situ hybridizations were performed as described, with minor modifications (Brand et al., 2002). For scanning electron microscopy, plant material was treated as described previously (Kwiatkowska, 2004). A Zeiss LSM510 was used for confocal microscopy, with settings recommended by the manufacturer.

Nuclear DNA Quantification

Trichomes were isolated from the Columbia wild type and induced JLO-GR plants 2 d after induction, following published protocols (Zhang and Oppenheimer, 2004). Pixel areas of nuclei stained with 1 μM YO-YO1 were measured with ImageJ and DISKUS (Leica) software. Since leaf cells can vary in their ploidy levels, so we used the generally diploid guard cell nuclei (2C) for calibration. For analysis of leaf nuclei, 35S:JLO-GR plants were first induced with Dex at 14 DAG. Leaves of different developmental stages were collected at 23 d after inductions started and analyzed as described (De Veylder et al., 2001). Leaves at the same developmental stage of Dex-treated Columbia (wild-type) plants served as controls.

Auxin Transport Measurement

Measurement of auxin transport was performed as described previously, with minor modifications (Bennett et al., 2006). Inflorescence stem segments (2.5 cm) from DR5rev:GFP or DR5rev:GFP;JLO-GR plants were cut and incubated in 30 μL of 50 mM HEPES, pH 7, and 1 mM CaCl2 containing 1 μCi of 3H-IAA in normal or inverted orientation for 18 h at room temperature. Segments (0.5 cm) from the exposed end of the shoot were then cut, and IAA was extracted for 48 h in 500 μL 70% methanol. Radioactivity was then quantified against a 3H standard in a scintillation counter.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative data library under accession numbers At4g00220 (JLO) and At4g00210 (LBD31).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Map of the JLO Gene and Insertion Mutants.
  • Supplemental Figure 2. Arrest of Seed Development in jlo-2 Mutants.
  • Supplemental Table 1. DR5rev:GFP Expression in Embryos.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Thomas Kirch for discovering the jlo-D mutant, Wolfgang Werr for donating the STM:GUS reporter line and collaboration in the TAMARA activation tagging project, Lieven de Veylder for support with nuclear sorting, Jiri Friml for kindly providing the DR5rev:GFP line, and Daniel Schubert for comments on the manuscript. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutant. Carin Theres and Rebecca Kloppenburg analyzed T-DNA insertion lines and provided technical support. Part of this work was supported by the Deutsch Forschungsgemeinschaft through SFB590 and the Arabidopsis Functional Genomics Network and by the European Union through a Marie Curie Research and Training Network (SY-STEM).

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Rüdiger Simon (ed.frodlesseud-inu@nomis.regideur).

[W]Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.106.047159

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