In plants the dorsoventral boundary of leaves defines an axis of symmetry through the centre of the organ separating the top (dorsal) and bottom (ventral) tissues. Although the positioning of this boundary is critical for leaf morphogenesis, how the boundary is established and how it influences development remains unclear. Using live-imaging and perturbation experiments we show that leaf orientation, morphology and position are pre-patterned by HD-ZIPIII and KAN gene expression in the shoot, leading to a model in which dorsoventral genes coordinate to regulate plant development by localizing auxin response between their expression domains. However we also find that auxin levels feedback on dorsoventral patterning by spatially organizing HD-ZIPIII and KAN expression in the shoot periphery. By demonstrating that the regulation of these genes by auxin also governs their response to wounds, our results also provide a parsimonious explanation for the influence of wounds on leaf dorsoventrality.

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial-abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets. T he morphogenesis of lateral organs in plants and animals is dependent on the specification of distinct cell types early in development. In particular, the correct patterning of adaxialabaxial tissues in plant organs such as leaves is critical for the generation of a lamina shape and the formation of a polar vascular system (1-4). Adaxial-abaxial cell-type patterning in turn depends on the restricted expression of several genes known to specify these cell types, including the class III homeodomain leucine zipper genes (HD-ZIPIIIs), KANADI genes, HD-ZIPIIs, and microRNA (MIR)165/166 (1, 2, 4-11). In general, genetic analyses have indicated that adaxial and abaxial factors act oppositely in organ patterning (1,2,4,(8)(9)(10)(11). Hence, loss-of-function mutations in genes promoting adaxial cell identity typically cause an abaxialized phenotype that correlates with the ectopic expression of abaxial genes, whereas loss-of-function mutations in abaxial genes produce an adaxialized phenotype that is accompanied by the expanded expression of adaxial genes. This antagonistic interaction between adaxial and abaxial factors may be mediated by mutually antagonistic regulation (12) or through opposing regulation of common targets (9, 13-16).A key set of transcription factors involved in plant organ polarity are the HD-ZIPIII proteins, such as REVOLUTA (REV), which specify adaxial cell fate (1, 2, 4, 17). The expression of these genes is restricted specifically to adaxial tissues via the action of two miRNA families, MIR165 and MIR166 (2, 7). In turn, the expression of these miRNAs is restricted to abaxial tissues and this restriction is essential for maintaining proper organ polarity (18).Here, we address the question of how MIR165/166 are regulated. We show that the HD-ZIPII proteins HAT3 and ATHB4 physically interact with HD-ZIPIII proteins and directly repress MIR165/166 expression via a conserved cis-element located in their promoters. This regulatory interaction largely accounts for HAT3 and ATHB4 function and reveals the molecular nature of a bidirectional repressive circuit essential to maintain balance between adaxial and abaxial tissue sp...

Abstract:In plants the dorsoventral boundary of leaves defines an axis of symmetry 22 through the centre of the organ separating the top (dorsal) and bottom (ventral) tissues. 23Although the positioning of this boundary is critical for leaf morphogenesis, how the 24 boundary is established and how it influences development remains unclear. Using live-25 imaging and perturbation experiments we show that leaf orientation, morphology and 26 position are pre-patterned by HD-ZIPIII and KAN gene expression in the shoot, leading 27 to a model in which dorsoventral genes coordinate to regulate plant development by 28 localizing auxin response between their expression domains. However we also find that 29 auxin levels feedback on dorsoventral patterning by spatially organizing HD-ZIPIII and 30

In the Arabidopsis thaliana shoot apical meristem (SAM) the expression domains of Class III Homeodomain Leucine Zipper (HD-ZIPIII) and KANADI (KAN) genes are separated by a narrow boundary region from which new organs are initiated. Disruption of this boundary through either loss of function or ectopic expression of HD-ZIPIII and KAN causes ectopic or suppression of organ formation respectively, raising the question of how these transcription factors regulate organogenesis at a molecular level. In this study we develop a multi-channel FACS/RNA-seq approach to characterize global patterns of gene expression across the HD-ZIPIII-KAN1 SAM boundary. We then combine FACS, RNA-seq and perturbations of HD-ZIPIII and KAN expression to identify genes that are both responsive to REV and KAN1 and normally expressed in patterns that correlate with REV and KAN1. Our data reveal that a significant number of genes responsive to REV are regulated in opposite ways depending on time after induction, with genes associated with auxin response and synthesis upregulated initially, but later repressed. We also characterize the cell type specific expression patterns of auxin responsive genes and identify a set of genes involved in organogenesis repressed by both REV and KAN1.

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