Conserved in a variety of evolutionarily divergent plant species, LOB DOMAIN (LBD) genes define a large, plant-specific family of largely unknown function. LBD genes have been implicated in a variety of developmental processes in plants, although to date, relatively few members have been assigned functions. LBD proteins have previously been predicted to be transcription factors, however supporting evidence has only been circumstantial. To address the biochemical function of LBD proteins, we identified a 6-bp consensus motif recognized by a wide cross-section of LBD proteins, and showed that LATERAL ORGAN BOUNDARIES (LOB), the founding member of the family, is a transcriptional activator in yeast. Thus, the LBD genes encode a novel class of DNA-binding transcription factors. Post-translational regulation of transcription factors is often crucial for control of gene expression. In our study, we demonstrate that members of the basic helix–loop–helix (bHLH) family of transcription factors are capable of interacting with LOB. The expression patterns of bHLH048 and LOB overlap at lateral organ boundaries. Interestingly, the interaction of bHLH048 with LOB results in reduced affinity of LOB for the consensus DNA motif. Thus, our studies suggest that bHLH048 post-translationally regulates the function of LOB at lateral organ boundaries.

Leaves and flowers begin life as outgrowths from the edges of shoot apical meristems. Stem cell divisions in the meristem center replenish cells that are incorporated into organ primordia at the meristem periphery and leave the meristem. Organ boundaries, regions of limited growth that separate forming organs from the meristem, serve to isolate these two domains and are critical for coordination of organogenesis and meristem maintenance. Boundary formation and maintenance are poorly understood processes, despite the identification of a number of boundary-specific transcription factors. Here we provide genetic and biochemical evidence that the Arabidopsis thaliana transcription factor LATERAL ORGAN BOUNDARIES (LOB) negatively regulates accumulation of the plant steroid hormone brassinosteroid (BR) in organ boundaries. We found that ectopic expression of LOB results in reduced BR responses. We identified BAS1, which encodes a BR-inactivating enzyme, as a direct target of LOB transcriptional activation. Loss-of-function lob mutants exhibit organ fusions, and this phenotype is suppressed by expression of BAS1 under the LOB promoter, indicating that BR hyperaccumulation contributes to the lob mutant phenotype. In addition, LOB expression is BR regulated; therefore, LOB and BR form a feedback loop to modulate local BR accumulation in organ boundaries to limit growth in the boundary domain.L eaves and flowers are produced from the periphery of the shoot apical meristem, a self-perpetuating structure containing a population of self-renewing stem cells. Stem cell divisions in the meristem center replenish the cells that are incorporated into organ primordia at the meristem periphery and exit the meristem (1). The balance between organogenesis and meristem maintenance is essential for continued organ formation, and the boundary between the meristem and organ primordia plays a key role in maintaining the integrity of the meristem and differentiating organs. Boundary cells are small and divide infrequently relative to cells in the adjacent regions; thus, the boundary is a discrete domain that is distinct from the meristem and organ primordia (2-4). During organ formation, inhibition of growth in the boundary allows formation of a cleft, which results in separation of the forming organ from the meristem. A number of boundary-specific transcription factors in several families act redundantly to specify organ boundary cell fate and meristem maintenance (5-11). Few targets of boundary-specific transcription factors have been identified, and little is known about the physiological and biochemical processes they regulate.Arabidopsis LATERAL ORGAN BOUNDARIES (LOB) encodes a member of the plant-specific LOB-domain transcription factor family and is expressed specifically in organ boundaries (12). To investigate the developmental function of LOB, we examined the consequence of increased and decreased LOB activity and used expression profiling to identify targets of LOB transcriptional regulation. We show that LOB negatively regulate...

The flattening of leaves results from the interaction between upper (adaxial) and lower (abaxial) domains in the developing primordium. These domains are specified by conserved, overlapping genetic pathways involving several distinct transcription factor families and small regulatory RNAs. Polarity determinants employ a series of antagonistic interactions to produce mutually exclusive cell fates whose positioning is likely refined by signaling across the adaxial–abaxial boundary. Signaling candidates include a mobile small RNA—the first positional signal described in adaxial–abaxial polarity. Possible mechanisms to polarize the incipient primordium are discussed, including meristem-derived signaling and a model in which a polarized organogenic zone prepatterns the adaxial–abaxial axis.

Small RNAs have emerged as a new class of mobile signals. Here, we investigate their mechanism of action and show that mobile small RNAs generate sharply defined domains of target gene expression through an intrinsic and direct threshold-based readout of their mobility gradients. This readout is highly sensitive to small RNA levels at the source, allowing plasticity in the positioning of a target gene expression boundary. Besides patterning their immediate targets, the readouts of opposing small RNA gradients enable specification of robust, uniformly positioned developmental boundaries. These patterning properties of small RNAs are reminiscent of those of animal morphogens. However, their mode of action and the intrinsic nature of their gradients distinguish mobile small RNAs from classical morphogens and present a unique direct mechanism through which to relay positional information. Mobile small RNAs and their targets thus emerge as highly portable, evolutionarily tractable regulatory modules through which to create pattern.

Flattened leaf architecture is not a default state but depends on positional information to precisely coordinate patterns of cell division in the growing primordium. This information is provided, in part, by the boundary between the adaxial (top) and abaxial (bottom) domains of the leaf, which are specified via an intricate gene regulatory network whose precise circuitry remains poorly defined. Here, we examined the contribution of the ASYMMETRIC LEAVES (AS) pathway to adaxial-abaxial patterning in Arabidopsis thaliana and demonstrate that AS1-AS2 affects this process via multiple, distinct regulatory mechanisms. AS1-AS2 uses Polycomb-dependent and -independent mechanisms to directly repress the abaxial determinants MIR166A, YABBY5, and AUXIN RESPONSE FACTOR3 (ARF3), as well as a nonrepressive mechanism in the regulation of the adaxial determinant TAS3A. These regulatory interactions, together with data from prior studies, lead to a model in which the sequential polarization of determinants, including AS1-AS2, explains the establishment and maintenance of adaxial-abaxial leaf polarity. Moreover, our analyses show that the shared repression of ARF3 by the AS and trans-acting small interfering RNA (ta-siRNA) pathways intersects with additional AS1-AS2 targets to affect multiple nodes in leaf development, impacting polarity as well as leaf complexity. These data illustrate the surprisingly multifaceted contribution of AS1-AS2 to leaf development showing that, in conjunction with the ta-siRNA pathway, AS1-AS2 keeps the Arabidopsis leaf both flat and simple.

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

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Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.