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Arabidopsis microRNA165a (miR165a) targets Class III Homeodomain Leucine-Zipper (HD-ZIPIII) transcription factors to regulate various aspects of plant development and stress response. Over-expression of miR165a mimics the loss-of-function phenotype of HD-ZIPIII genes and leading to ectopic organ formation, shoot apical meristem (SAM) termination, loss of leaf polarity, and defective vasculature development. However, the molecular mechanisms underlying these phenotypes remain unresolved. Here, we over-expressed miR165a in a dexamethasone inducible manner and identified differentially expressed genes in the SAM through RNA-Seq. Simultaneously, using multi-channel FACS combined with RNA-Seq approach, we characterized global transcriptome patterns in miR165a expressing cell-types compared to HD-ZIPIII expressing cell-types and other cell-types in SAM. By integrating our results we identified sets of genes which are up-regulated by miR165a as well have enriched expression in miR165a cell-types, and vice-versa. Known plant development related genes such as HD-ZIPIII and their targets LITTLE ZIPPERs, Like AUXIN RESISTANT 2, BEL1-like homeodomain 6, ROTUNDIFOLIA like 16 were found to be down-regulated. Among the up-regulated genes, GIBBERELLIN 2-OXIDASEs, various elemental transporters (YSL3, ZIFL1, SULTR), and other transporter genes were prominent. Thus, the genes identified in this study help to unravel the molecular mechanism of miR165a and HD-ZIPIII regulated plant development and stress response., (© 2023. The Author(s).)

Plant development is regulated by transcription factors that often act in more than one process and stage of development. Yet the molecular mechanisms that govern the functional diversity and specificity of these proteins remains far from understood. Flower development provides an ideal context to study these mechanisms since the development of distinct floral organs depends on similar but distinct combinations of transcriptional regulators. Recent work also highlights the importance of leaf polarity regulators as additional key factors in flower initiation, floral organ morphogenesis, and possibly floral organ positioning. A detailed understanding of how these factors work in combination will enable us to address outstanding questions in flower development including how distinct shapes and positions of floral organs are generated. Experimental approaches and computer-based modeling will be required to characterize gene-regulatory networks at the level of single cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cellular heterogeneity is a hallmark of multicellular organisms. During shoot regeneration from undifferentiated callus, only a select few cells, called progenitors, develop into shoot. How these cells are selected and what governs their subsequent progression to a patterned organ system is unknown. Using Arabidopsis thaliana, we show that it is not just the abundance of stem cell regulators but rather the localization pattern of polarity proteins that predicts the progenitor's fate. A shoot-promoting factor, CUC2, activated the expression of the cell-wall-loosening enzyme, XTH9, solely in a shell of cells surrounding the progenitor, causing different mechanical stresses in these cells. This mechanical conflict then activates cell polarity in progenitors to promote meristem formation. Interestingly, genetic or physical perturbations to cells surrounding the progenitor impaired the progenitor and vice versa. These suggest a feedback loop between progenitors and their neighbors for shoot regeneration in the absence of tissue-patterning cues., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

At the Arabidopsis shoot apex, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis. While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined. Here, we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighboring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighboring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Over the last decade or so important progress has been made in identifying and understanding a set of patterning mechanisms that have the potential to explain many aspects of plant morphology. These include the feedback loop between mechanical stresses and interphase microtubules, the regulation of plant cell polarity and the role of adaxial and abaxial cell type boundaries. What is perhaps most intriguing is how these mechanisms integrate in a combinatorial manner that provides a means to generate a large variety of commonly seen plant morphologies. Here, I review our current understanding of these mechanisms and discuss the links between them., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Heisler.)

Plants develop lateral organs such as leaves and flowers throughout their post-embryonic life from a structure called the shoot apical meristem (SAM), located at the plant shoot apex. This process is highly dynamic, and therefore in order to understand meristem and organ development, it is critical to be able to analyze these processes with high temporal and spatial resolution. Although several protocols have been published for imaging the Arabidopsis inflorescence meristem, gaining access to the vegetative meristem for imaging has been considered more difficult. Here we describe a method to dissect young Arabidopsis seedlings in order to obtain a clear view of the vegetative meristem and young leaf primordia using confocal microscopy.

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., Competing Interests: The authors have declared that no competing interests exist.

Plants continuously produce lateral organs from the shoot apex such as leaves and flowers, providing an excellent opportunity to study their development. The plant hormone auxin plays a central role in this process by promoting organ formation where it accumulates due to polar auxin transport. Recently, the use of live-imaging, fine perturbation techniques and computational modelling has helped researchers make exciting progress in addressing long-standing questions on plant organogenesis, not only regarding the role of auxin in promoting growth but also on the regulation of morphogenesis and transcriptional control. In this review, we discuss a number of recent studies that address these points, with particular reference to how auxin acts in early leaf development and in leaf shape., (Copyright © 2019. Published by Elsevier Ltd.)

In plants mechanical signals pattern morphogenesis through the polar transport of the hormone auxin and through regulation of interphase microtubule (MT) orientation. To date, the mechanisms by which such signals induce changes in cell polarity remain unknown. Through a combination of time-lapse imaging, and chemical and mechanical perturbations, we show that mechanical stimulation of the SAM causes transient changes in cytoplasmic calcium ion concentration (Ca 2+ ) and that transient Ca 2+ response is required for downstream changes in PIN-FORMED 1 (PIN1) polarity. We also find that dynamic changes in Ca 2+ occur during development of the SAM and this Ca 2+ response is required for changes in PIN1 polarity, though not sufficient. In contrast, we find that Ca 2+ is not necessary for the response of MTs to mechanical perturbations revealing that Ca 2+ specifically acts downstream of mechanics to regulate PIN1 polarity response.

Dorsoventrality in leaves has been shown to depend on the pre-patterned expression of KANADI and HD-ZIPIII genes within the plant shoot apical meristem (SAM). However, it has also been proposed that asymmetric auxin levels within initiating leaves help establish leaf polarity, based in part on observations of the DII auxin sensor. By analyzing and quantifying the expression of the R2D2 auxin sensor, we find that there is no obvious asymmetry in auxin levels during Arabidopsis leaf development. We further show that the mDII control sensor also exhibits an asymmetry in expression in developing leaf primordia early on, while it becomes more symmetric at a later developmental stage as reported previously. Together with other recent findings, our results argue against the importance of auxin asymmetry in establishing leaf polarity., Competing Interests: NB, HÅ, HJ, MH No competing interests declared, (© 2019, Bhatia et al.)

Mutual interactions of the phytohormones, cytokinins and auxin determine root or shoot identity during postembryonic de novo organogenesis in plants. However, our understanding of the role of hormonal metabolism and perception during early stages of cell fate reprogramming is still elusive. Here we show that auxin activates root formation, whereas cytokinins mediate early loss of the root identity, primordia disorganisation and initiation of shoot development. Exogenous and endogenous cytokinins influence the initiation of newly formed organs, as well as the pace of organ development. The process of de novo shoot apical meristem establishment is accompanied by accumulation of endogenous cytokinins, differential regulation of genes for individual cytokinin receptors, strong activation of AHK4-mediated signalling and induction of the shoot-specific homeodomain regulator WUSCHEL. The last is associated with upregulation of isopentenyladenine-type cytokinins, revealing higher shoot-forming potential when compared with trans -zeatin. Moreover, AHK4-controlled cytokinin signalling negatively regulates the root stem cell organiser WUSCHEL RELATED HOMEOBOX 5 in the root quiescent centre. We propose an important role for endogenous cytokinin biosynthesis and AHK4-mediated cytokinin signalling in the control of de novo -induced organ identity., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Periodic patterns during development often occur spontaneously through a process of self-organization. While reaction-diffusion mechanisms are often invoked, other types of mechanisms that involve cell-cell interactions and mechanical buckling have also been identified. Phyllotaxis, or the positioning of plant organs, has emerged as an excellent model system to study the self-organization of periodic patterns. At the macro scale, the regular spacing of organs on the growing plant shoot gives rise to the typical spiral and whorled arrangements of plant organs found in nature. In turn, this spacing relies on complex patterns of cell polarity that involve feedback between a signaling molecule - the plant hormone auxin - and its polar, cell-to-cell transport. Here, we review recent progress in understanding phyllotaxis and plant cell polarity and highlight the development of new tools that can help address the remaining gaps in our understanding., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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.

Leaves are present in all land plants and are specialized organs for light harvesting. They arise at the flanks of the shoot apical meristem (SAM), and develop into lamina structures that exhibit adaxial/abaxial (upper/lower side of the leaf) polarity. At the molecular level, an intricate regulatory network determines ad-/abaxial polarity in Arabidopsis thaliana leaves, where the Class III Homeodomain Leucine Zipper (HD-ZIPIII) and KANADI (KAN) proteins are key mediators. The HD-ZIPIII REVOLUTA (REV) is expressed in the adaxial domain of lateral organs, whereas KAN1 is involved in abaxial differentiation. The REV/KAN1 module directly and antagonistically regulates the expression of several genes involved in shade-induced growth and auxin biosynthetic enzymes., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

The periodic formation of plant organs such as leaves and flowers gives rise to intricate patterns that have fascinated biologists and mathematicians alike for hundreds of years [1]. The plant hormone auxin plays a central role in establishing these patterns by promoting organ formation at sites where it accumulates due to its polar, cell-to-cell transport [2-6]. Although experimental evidence as well as modeling suggest that feedback from auxin to its transport direction may help specify phyllotactic patterns [7-12], the nature of this feedback remains unclear [13]. Here we reveal that polarization of the auxin efflux carrier PIN-FORMED 1 (PIN1) is regulated by the auxin response transcription factor MONOPTEROS (MP) [14]. We find that in the shoot, cell polarity patterns follow MP expression, which in turn follows auxin distribution patterns. By perturbing MP activity both globally and locally, we show that localized MP activity is necessary for the generation of polarity convergence patterns and that localized MP expression is sufficient to instruct PIN1 polarity directions non-cell autonomously, toward MP-expressing cells. By expressing MP in the epidermis of mp mutants, we further show that although MP activity in a single-cell layer is sufficient to promote polarity convergence patterns, MP in sub-epidermal tissues helps anchor these polarity patterns to the underlying cells. Overall, our findings reveal a patterning module in plants that determines organ position by orienting transport of the hormone auxin toward cells with high levels of MP-mediated auxin signaling. We propose that this feedback process acts broadly to generate periodic plant architectures., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Shoot branching requires the establishment of new meristems harboring stem cells; this phenomenon raises questions about the precise regulation of meristematic fate. In seed plants, these new meristems initiate in leaf axils to enable lateral shoot branching. Using live-cell imaging of leaf axil cells, we show that the initiation of axillary meristems requires a meristematic cell population continuously expressing the meristem marker SHOOT MERISTEMLESS (STM). The maintenance of STM expression depends on the leaf axil auxin minimum. Ectopic expression of STM is insufficient to activate axillary buds formation from plants that have lost leaf axil STM expressing cells. This suggests that some cells undergo irreversible commitment to a developmental fate. In more mature leaves, REVOLUTA (REV) directly up-regulates STM expression in leaf axil meristematic cells, but not in differentiated cells, to establish axillary meristems. Cell type-specific binding of REV to the STM region correlates with epigenetic modifications. Our data favor a threshold model for axillary meristem initiation, in which low levels of STM maintain meristematic competence and high levels of STM lead to meristem initiation.

Two interrelated problems in biology are understanding the regulatory logic and predictability of morphological evolution. Here, we studied these problems by comparing Arabidopsis thaliana, which has simple leaves, and its relative, Cardamine hirsuta, which has dissected leaves comprising leaflets. By transferring genes between the two species, we provide evidence for an inverse relationship between the pleiotropy of SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) homeobox genes and their ability to modify leaf form. We further show that cis-regulatory divergence of BP results in two alternative configurations of the genetic networks controlling leaf development. In C. hirsuta, ChBP is repressed by the microRNA164A (MIR164A)/ChCUP-SHAPED COTYLEDON (ChCUC) module and ChASYMMETRIC LEAVES1 (ChAS1), thus creating cross-talk between MIR164A/CUC and AS1 that does not occur in A. thaliana. These different genetic architectures lead to divergent interactions of network components and growth regulation in each species. We suggest that certain regulatory genes with low pleiotropy are predisposed to readily integrate into or disengage from conserved genetic networks influencing organ geometry, thus rapidly altering their properties and contributing to morphological divergence., (© 2015 Rast-Somssich et al.; Published by Cold Spring Harbor Laboratory Press.)

The aboveground tissues of higher plants are derived from a small population of stem cells located at the shoot apex within a structure called the shoot apical meristem (SAM). The SAM not only includes the stem cells but also incorporates a region from which lateral organs arise. The SAM is therefore of prime interest for understanding plant growth and development. In this chapter we outline methods for using confocal microscopy to image the Arabidopsis inflorescence SAM. This method enables detailed examination of cell division and growth patterns (Reddy et al., Development 131:4225-4237, 2004) as well as gene expression and protein localization patterns over time (Heisler et al. Curr Biol 15:1899-1911, 2005). When combined with perturbation approaches, the method offers an extremely powerful system for investigating SAM function in great detail.

Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.

Plant organ development and polarity establishment is mediated by the action of several transcription factors. Among these, the KANADI (KAN) subclade of the GARP protein family plays important roles in polarity-associated processes during embryo, shoot and root patterning. In this study, we have identified a set of potential direct target genes of KAN1 through a combination of chromatin immunoprecipitation/DNA sequencing (ChIP-Seq) and genome-wide transcriptional profiling using tiling arrays. Target genes are over-represented for genes involved in the regulation of organ development as well as in the response to auxin. KAN1 affects directly the expression of several genes previously shown to be important in the establishment of polarity during lateral organ and vascular tissue development. We also show that KAN1 controls through its target genes auxin effects on organ development at different levels: transport and its regulation, and signaling. In addition, KAN1 regulates genes involved in the response to abscisic acid, jasmonic acid, brassinosteroids, ethylene, cytokinins and gibberellins. The role of KAN1 in organ polarity is antagonized by HD-ZIPIII transcription factors, including REVOLUTA (REV). A comparison of their target genes reveals that the REV/KAN1 module acts in organ patterning through opposite regulation of shared targets. Evidence of mutual repression between closely related family members is also shown.

Development is a dynamic process occurring at the microscopic scale. The ability to see how it unfolds in detail is invaluable not only for helping us appreciate its full complexity but also to experimentally dissect its mechanisms. The sophistication of experimental approaches and imaging technologies has increased over the past decade at an astounding pace. In this review we highlight and discuss several studies that illustrate the latest advances in the application of live-imaging to dissect plant development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The transcription factor WUSCHEL (WUS) acts from a well-defined domain within the Arabidopsis thaliana shoot apical meristem (SAM) to maintain a stem cell niche. A negative-feedback loop involving the CLAVATA (CLV) signaling pathway regulates the number of WUS-expressing cells and provides the current paradigm for the homeostatic maintenance of stem cell numbers. Despite the continual turnover of cells in the SAM during development, the WUS domain remains patterned at a fixed distance below the shoot apex. Recent work has uncovered a positive-feedback loop between WUS function and the plant hormone cytokinin. Furthermore, loss of function of the cytokinin biosynthetic gene, LONELY GUY (LOG), results in a wus-like phenotype in rice. Herein, we find the Arabidopsis LOG4 gene is expressed in the SAM epidermis. We use this to develop a computational model representing a growing SAM to suggest the plausibility that apically derived cytokinin and CLV signaling, together, act as positional cues for patterning the WUS domain within the stem cell niche. Furthermore, model simulations backed by experimental data suggest a previously unknown negative feedback between WUS function and cytokinin biosynthesis in the Arabidopsis SAM epidermis. These results suggest a plausible dynamic feedback principle by which the SAM stem cell niche is patterned.

Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis., Competing Interests: The authors have declared that no competing interests exist.

A central question in developmental biology is whether and how mechanical forces serve as cues for cellular behavior and thereby regulate morphogenesis. We found that morphogenesis at the Arabidopsis shoot apex depends on the microtubule cytoskeleton, which in turn is regulated by mechanical stress. A combination of experiments and modeling shows that a feedback loop encompassing tissue morphology, stress patterns, and microtubule-mediated cellular properties is sufficient to account for the coordinated patterns of microtubule arrays observed in epidermal cells, as well as for patterns of apical morphogenesis.

Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.

In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.

Meristems continually supply new cells for post-embryonic plant development and coordinate the initiation of new organs, such as leaves and flowers. Meristem function is regulated by a large and interconnected dynamic system that includes transcription networks, intercellular protein signalling, polarized transport of hormones and a constantly changing cellular topology. Mathematical modelling, in which the dynamics of a system are simulated using explicitly defined interactions, can serve as a powerful tool for examining the expected behaviour of such a system given our present knowledge and assumptions. Modelling can also help to investigate new hypotheses in silico both to validate ideas and to obtain inspiration for new experiments. Several recent studies have used new molecular data together with modelling and computational techniques to investigate meristem function.

Recent studies show that plant organ positioning may be mediated by localized concentrations of the plant hormone auxin. Auxin patterning in the shoot apical meristem is in turn brought about by the subcellular polar distribution of the putative auxin efflux mediator, PIN1. However, the question of what signals determine PIN1 polarization and how this gives rise to regular patterns of auxin concentration remains unknown. Here we address these questions by using mathematical modeling combined with confocal imaging. We propose a model that is based on the assumption that auxin influences the polarization of its own efflux within the meristem epidermis. We show that such a model is sufficient to create regular spatial patterns of auxin concentration on systems with static and dynamic cellular connectivities, the latter governed by a mechanical model. We also optimize parameter values for the PIN1 dynamics by using a detailed auxin transport model, for which parameter values are taken from experimental estimates, together with a template consisting of cell and wall compartments as well as PIN1 concentrations quantitatively extracted from confocal data. The model shows how polarized transport can drive the formation of regular patterns.

Plant biology is currently confronted with an overflow of expression profile data provided by high-throughput microarray transcription analyses. However, the tissue and cellular resolution of these techniques is limited. Thus, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here we present an in situ mRNA hybridization method that is routinely used in the analysis of gene expression patterns. The protocol is optimized for mRNA localizations in sectioned tissue of Arabidopsis seedlings including embryos, roots, hypocotyls, young primary leaves and flowers. The detailed protocol, recommended controls and troubleshooting are presented along with examples of application. The total time for the process is 10 days.

Background: Plants produce leaf and flower primordia from a specialized tissue called the shoot apical meristem (SAM). Genetic studies have identified a large number of genes that affect various aspects of primordium development including positioning, growth, and differentiation. So far, however, a detailed understanding of the spatio-temporal sequence of events leading to primordium development has not been established., Results: We use confocal imaging of green fluorescent protein (GFP) reporter genes in living plants to monitor the expression patterns of multiple proteins and genes involved in flower primordial developmental processes. By monitoring the expression and polarity of PINFORMED1 (PIN1), the auxin efflux facilitator, and the expression of the auxin-responsive reporter DR5, we reveal stereotypical PIN1 polarity changes which, together with auxin induction experiments, suggest that cycles of auxin build-up and depletion accompany, and may direct, different stages of primordium development. Imaging of multiple GFP-protein fusions shows that these dynamics also correlate with the specification of primordial boundary domains, organ polarity axes, and the sites of floral meristem initiation., Conclusions: These results provide new insight into auxin transport dynamics during primordial positioning and suggest a role for auxin transport in influencing primordial cell type.

Precise knowledge of spatial and temporal patterns of cell division, including number and orientation of divisions, and knowledge of cell expansion, is central to understanding morphogenesis. Our current knowledge of cell division patterns during plant and animal morphogenesis is largely deduced from analysis of clonal shapes and sizes. But such an analysis can reveal only the number, not the orientation or exact rate, of cell divisions. In this study, we have analyzed growth in real time by monitoring individual cell divisions in the shoot apical meristems (SAMs) of Arabidopsis thaliana. The live imaging technique has led to the development of a spatial and temporal map of cell division patterns. We have integrated cell behavior over time to visualize growth. Our analysis reveals temporal variation in mitotic activity and the cell division is coordinated across clonally distinct layers of cells. Temporal variation in mitotic activity is not correlated to the estimated plastochron length and diurnal rhythms. Cell division rates vary across the SAM surface. Cells in the peripheral zone (PZ) divide at a faster rate than in the central zone (CZ). Cell division rates in the CZ are relatively heterogeneous when compared with PZ cells. We have analyzed the cell behavior associated with flower primordium development starting from a stage at which the future flower comprises four cells in the L1 epidermal layer. Primordium development is a sequential process linked to distinct cellular behavior. Oriented cell divisions, in primordial progenitors and in cells located proximal to them, are associated with initial primordial outgrowth. The oriented cell divisions are followed by a rapid burst of cell expansion and cell division, which transforms a flower primordium into a three-dimensional flower bud. Distinct lack of cell expansion is seen in a narrow band of cells, which forms the boundary region between developing flower bud and the SAM. We discuss these results in the context of SAM morphogenesis.

Important goals in understanding leaf development are to identify genes involved in pattern specification, and also genes that translate this information into cell types and tissue structure. Loss-of-function mutations at the JAGGED (JAG) locus result in Arabidopsis plants with abnormally shaped lateral organs including serrated leaves, narrow floral organs, and petals that contain fewer but more elongate cells. jag mutations also suppress bract formation in leafy, apetala1 and apetala2 mutant backgrounds. The JAG gene was identified by map-based cloning to be a member of the zinc finger family of plant transcription factors and encodes a protein similar in structure to SUPERMAN with a single C(2)H(2)-type zinc finger, a proline-rich motif and a short leucine-rich repressor motif. JAG mRNA is localized to lateral organ primordia throughout the plant but is not found in the shoot apical meristem. Misexpression of JAG results in leaf fusion and the development of ectopic leaf-like outgrowth from both vegetative and floral tissues. Thus, JAG is necessary for proper lateral organ shape and is sufficient to induce the proliferation of lateral organ tissue.

Studies involving mutants of the gene SPATULA indicate that it promotes the growth of carpel margins and of pollen tract tissues derived from them. We show that it encodes a new member of the basic-helix-loop-helix family of transcription factors. SPATULA is expressed in marginal and pollen tract tissues throughout their development confirming its role in regulating their growth. It is also expressed in many other tissues where it may act redundantly to control growth, including the peripheral zone of the shoot apical meristem, and specific tissues within leaves, petals, stamens and roots. Expression in the stomium, funiculus and valve dehiscence zone indicates an additional role in abscission. SPATULA expression does not require the function of the other carpel development genes CRABS CLAW and AGAMOUS, although its expression is repressed in first whorl organs by the A function gene APETALA2. Further, we have shown that disruptions to gynoecial pattern formation seen in ettin mutants can largely be attributed to ectopic SPATULA action. ETTIN's role seems to be to negatively regulate SPATULA expression in abaxial regions of the developing gynoecium. SPATULA is the first basic-helix-loop-helix gene in plants known to play a role in floral organogenesis.