Monoclonal Antibody Disrupts Biofilm Structure and Restores Antibiotic Susceptibility in an Orthopedic Implant Infection Model.

Bacterial biofilms on orthopedic implants are resistant to the host immune response and to traditional systemic antibiotics. Novel therapies are needed to improve patient outcomes. TRL1068 is a human monoclonal antibody (mAb) against a biofilm anchoring protein. For assessment of this agent in an orthopedic implant infection model, efficacy was measured by reduction in bacterial burden of Staphylococcus aureus, the most common pathogen for prosthetic joint infections (PJI). Systemic treatment with the biofilm disrupting mAb TRL1068 in conjunction with vancomycin eradicated S. aureus from steel pins implanted in the spine for 26 of 27 mice, significantly more than for vancomycin alone. The mechanism of action was elucidated by two microscopy studies. First, TRL1068 was localized to biofilm using a fluorescent antibody tag. Second, a qualitative effect on biofilm structure was observed using scanning electron microscopy (SEM) to examine steel pins that had been treated in vivo. SEM images of implants retrieved from control mice showed abundant three-dimensional biofilms, whereas those from mice treated with TRL1068 did not. Clinical Significance: TRL1068 binds at high affinity to S. aureus biofilms, thereby disrupting the three-dimensional structure and significantly reducing implant CFUs in a well-characterized orthopedic model for which prior tested agents have shown only partial efficacy. TRL1068 represents a promising systemic treatment for orthopedic implant infection.

Expression of KNUCKLES in the Stem Cell Domain Is Required for Its Function in the Control of Floral Meristem Activity in Arabidopsis.

In the model plant Arabidopsis thaliana, the zinc-finger transcription factor KNUCKLES (KNU) plays an important role in the termination of floral meristem activity, a process that is crucial for preventing the overgrowth of flowers. The KNU gene is activated in floral meristems by the floral organ identity factor AGAMOUS (AG), and it has been shown that both AG and KNU act in floral meristem control by directly repressing the stem cell regulator WUSCHEL (WUS), which leads to a loss of stem cell activity. When we re-examined the expression pattern of KNU in floral meristems, we found that KNU is expressed throughout the center of floral meristems, which includes, but is considerably broader than the WUS expression domain. We therefore hypothesized that KNU may have additional functions in the control of floral meristem activity. To test this, we employed a gene perturbation approach and knocked down KNU activity at different times and in different domains of the floral meristem. In these experiments we found that early expression in the stem cell domain, which is characterized by the expression of the key meristem regulatory gene CLAVATA3 (CLV3), is crucial for the establishment of KNU expression. The results of additional genetic and molecular analyses suggest that KNU represses floral meristem activity to a large extent by acting on CLV3. Thus, KNU might need to suppress the expression of several meristem regulators to terminate floral meristem activity efficiently.

Cauliflower fractal forms arise from perturbations of floral gene networks.

Throughout development, plant meristems regularly produce organs in defined spiral, opposite, or whorl patterns. Cauliflowers present an unusual organ arrangement with a multitude of spirals nested over a wide range of scales. How such a fractal, self-similar organization emerges from developmental mechanisms has remained elusive. Combining experimental analyses in an Arabidopsis thaliana cauliflower-like mutant with modeling, we found that curd self-similarity arises because the meristems fail to form flowers but keep the "memory" of their transient passage in a floral state. Additional mutations affecting meristem growth can induce the production of conical structures reminiscent of the conspicuous fractal Romanesco shape. This study reveals how fractal-like forms may emerge from the combination of key, defined perturbations of floral developmental programs and growth dynamics.

Broadening the impact of plant science through innovative, integrative, and inclusive outreach.

Population growth and climate change will impact food security and potentially exacerbate the environmental toll that agriculture has taken on our planet. These existential concerns demand that a passionate, interdisciplinary, and diverse community of plant science professionals is trained during the 21st century. Furthermore, societal trends that question the importance of science and expert knowledge highlight the need to better communicate the value of rigorous fundamental scientific exploration. Engaging students and the general public in the wonder of plants, and science in general, requires renewed efforts that take advantage of advances in technology and new models of funding and knowledge dissemination. In November 2018, funded by the National Science Foundation through the Arabidopsis Research and Training for the 21st century (ART 21) research coordination network, a symposium and workshop were held that included a diverse panel of students, scientists, educators, and administrators from across the US. The purpose of the workshop was to re-envision how outreach programs are funded, evaluated, acknowledged, and shared within the plant science community. One key objective was to generate a roadmap for future efforts. We hope that this document will serve as such, by providing a comprehensive resource for students and young faculty interested in developing effective outreach programs. We also anticipate that this document will guide the formation of community partnerships to scale up currently successful outreach programs, and lead to the design of future programs that effectively engage with a more diverse student body and citizenry.

A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control.

We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development.

Imaging flowers: a guide to current microscopy and tomography techniques to study flower development.

Developmental biology relies heavily on our ability to generate three-dimensional images of live biological specimens through time, and to map gene expression and hormone response in these specimens as they undergo development. The last two decades have seen an explosion of new bioimaging technologies that have pushed the limits of spatial and temporal resolution and provided biologists with invaluable new tools. However, plant tissues are difficult to image, and no single technology fits all purposes; choosing between many bioimaging techniques is not trivial. Here, we review modern light microscopy and computed projection tomography methods, their capabilities and limitations, and we discuss their current and potential applications to the study of flower development and fertilization.

Visualization of Protein Coding, Long Noncoding, and Nuclear RNAs by Fluorescence in Situ Hybridization in Sections of Shoot Apical Meristems and Developing Flowers.

In addition to transcriptional regulation, gene expression is further modulated through mRNA spatiotemporal distribution, by RNA movement between cells, and by RNA localization within cells. Here, we have adapted RNA fluorescence in situ hybridization (FISH) to explore RNA localization in Arabidopsis (Arabidopsis thaliana). We show that RNA FISH on sectioned material can be applied to investigate the tissue and subcellular localization of meristem and flower development genes, cell cycle transcripts, and plant long noncoding RNAs. We also developed double RNA FISH to dissect the coexpression of different mRNAs at the shoot apex and nuclear-cytoplasmic separation of cell cycle gene transcripts in dividing cells. By coupling RNA FISH with fluorescence immunocytochemistry, we further demonstrate that a gene's mRNA and protein may be simultaneously detected, for example revealing uniform distribution of PIN-FORMED1 (PIN1) mRNA and polar localization of PIN1 protein in the same cells. Therefore, our method enables the visualization of gene expression at both transcriptional and translational levels with subcellular spatial resolution, opening up the possibility of systematically tracking the dynamics of RNA molecules and their cognate proteins in plant cells.

SUPERMAN regulates floral whorl boundaries through control of auxin biosynthesis.

Proper floral patterning, including the number and position of floral organs in most plant species, is tightly controlled by the precise regulation of the persistence and size of floral meristems (FMs). In Arabidopsis, two known feedback pathways, one composed of WUSCHEL (WUS) and CLAVATA3 (CLV3) and the other composed of AGAMOUS (AG) and WUS, spatially and temporally control floral stem cells, respectively. However, mounting evidence suggests that other factors, including phytohormones, are also involved in floral meristem regulation. Here, we show that the boundary gene SUPERMAN (SUP) bridges floral organogenesis and floral meristem determinacy in another pathway that involves auxin signaling. SUP interacts with components of polycomb repressive complex 2 (PRC2) and fine-tunes local auxin signaling by negatively regulating the expression of the auxin biosynthesis genes YUCCA1/4 (YUC1/4). In sup mutants, derepressed local YUC1/4 activity elevates auxin levels at the boundary between whorls 3 and 4, which leads to an increase in the number and the prolonged maintenance of floral stem cells, and consequently an increase in the number of reproductive organs. Our work presents a new floral meristem regulatory mechanism, in which SUP, a boundary gene, coordinates floral organogenesis and floral meristem size through fine-tuning auxin biosynthesis.

SUPERMAN prevents class B gene expression and promotes stem cell termination in the fourth whorl of Arabidopsis thaliana flowers.

The molecular and genetic networks underlying the determination of floral organ identity are well studied, but much less is known about how the flower is partitioned into four developmentally distinct whorls. The SUPERMAN gene is required for proper specification of the boundary between stamens in whorl 3 and carpels in whorl 4, as superman mutants exhibit supernumerary stamens but usually lack carpels. However, it has remained unclear whether extra stamens in superman mutants originate from an organ identity change in whorl 4 or the overproliferation of whorl 3. Using live confocal imaging, we show that the extra stamens in superman mutants arise from cells in whorl 4, which change their fate from female to male, while floral stem cells proliferate longer, allowing for the production of additional stamens.

Live Confocal Imaging of Developing Arabidopsis Flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Genetics and plant development.

There are only three grand theories in biology: the theory of the cell, the theory of the gene, and the theory of evolution. Two of these, the cell and gene theories, originated in the study of plants, with the third resulting in part from botanical considerations as well. Mendel's elucidation of the rules of inheritance was a result of his experiments on peas. The rediscovery of Mendel's work in 1900 was by the botanists de Vries, Correns, and Tschermak. It was only in subsequent years that animals were also shown to have segregation of genetic elements in the exact same manner as had been shown in plants. The story of developmental biology is different - while the development of plants has long been studied, the experimental and genetic approaches to developmental mechanism were developed via experiments on animals, and the importance of genes in development (e.g., Waddington, 1940) and their use for understanding developmental mechanisms came to botanical science much later - as late as the 1980s.

Live confocal imaging of Arabidopsis flower buds.

Recent advances in confocal microscopy, coupled with the development of numerous fluorescent reporters, provide us with a powerful tool to study the development of plants. Live confocal imaging has been used extensively to further our understanding of the mechanisms underlying the formation of roots, shoots and leaves. However, it has not been widely applied to flowers, partly because of specific challenges associated with the imaging of flower buds. Here, we describe how to prepare and grow shoot apices of Arabidopsis in vitro, to perform both single-point and time-lapse imaging of live, developing flower buds with either an upright or an inverted confocal microscope.

SQUINT promotes stem cell homeostasis and floral meristem termination in Arabidopsis through APETALA2 and CLAVATA signalling.

Plant meristems harbour stem cells, which allow for the continuous production of new organs. Here, an analysis of the role of SQUINT (SQN) in stem cell dynamics in Arabidopsis is reported. A close examination of sqn mutants reveals defects that are very similar to that of weak clavata (clv) mutants, both in the flower meristem (increased number of floral organs, occasional delay in stem cell termination) and in the shoot apical meristem (meristem and central zone enlargement, occasional fasciation). sqn has a very mild effect in a clv mutant background, suggesting that SQN and the CLV genes act in the same genetic pathway. Accordingly, a loss-of-function allele of SQN strongly rescues the meristem abortion phenotype of plants that overexpress CLV3. Altogether, these data suggest that SQN is necessary for proper CLV signalling. SQN was shown to be required for normal accumulation of various miRNAs, including miR172. One of the targets of miR172, APETALA2 (AP2), antagonizes CLV signalling. The ap2-2 mutation strongly suppresses the meristem phenotypes of sqn, indicating that the effect of SQN on stem cell dynamics is largely, but not fully, mediated by the miR172/AP2 tandem. This study refines understanding of the intricate genetic networks that control both stem cell homeostasis and floral stem cell termination, two processes that are critical for the proper development and fertility of the plant.

Flower development in Arabidopsis: there is more to it than learning your ABCs.

The field of Arabidopsis flower development began in the early 1980s with the initial description of several mutants including apetala1, apetala2, and agamous that altered floral organ identity (Koornneef and van der Veen, Theor Appl Genet 58:257-263, 1980; Koornneef et al., J Hered 74:265-272, 1983). By the end of the 1980s, these mutants were receiving more focused attention to determine precisely how they affected flower development (Komaki et al., Development 104:195-203, 1988; Bowman et al., Plant Cell 1:37-52, 1989). In the last quarter century, impressive progress has been made in characterizing the gene products and molecular mechanisms that control the key events in flower development. In this review, we briefly summarize the highlights of work from the past 25 years but focus on advances in the field in the last several years.

Flower development: open questions and future directions.

Almost three decades of genetic and molecular analyses have resulted in detailed insights into many of the processes that take place during flower development and in the identification of a large number of key regulatory genes that control these processes. Despite this impressive progress, many questions about how flower development is controlled in different angiosperm species remain unanswered. In this chapter, we discuss some of these open questions and the experimental strategies with which they could be addressed. Specifically, we focus on the areas of floral meristem development and patterning, floral organ specification and differentiation, as well as on the molecular mechanisms underlying the evolutionary changes that have led to the astounding variations in flower size and architecture among extant and extinct angiosperms.

REBELOTE, SQUINT, and ULTRAPETALA1 function redundantly in the temporal regulation of floral meristem termination in Arabidopsis thaliana.

In Arabidopsis thaliana, flowers are determinate, showing a fixed number of whorls. Here, we report on three independent genes, a novel gene REBELOTE (RBL; protein of unknown function), SQUINT (SQN; a cyclophilin), and ULTRAPETALA1 (ULT1; a putative transcription factor) that redundantly influence floral meristem (FM) termination. Their mutations, combined with each other or with crabs claw, the genetic background in which they were isolated, trigger a strong FM indeterminacy with reiterations of extra floral whorls in the center of the flower. The range of phenotypes suggests that, in Arabidopsis, FM termination is initiated from stages 3 to 4 onwards and needs to be maintained through stage 6 and beyond, and that RBL, SQN, and ULT1 are required for this continuous regulation. We show that mutant phenotypes result from a decrease of AGAMOUS (AG) expression in an inner 4th whorl subdomain. However, the defect of AG activity alone does not explain all reported phenotypes, and our genetic data suggest that RBL, SQN, and, to a lesser extent, ULT1 also influence SUPERMAN activity. Finally, from all the molecular and genetic data presented, we argue that these genes contribute to the more stable and uniform development of flowers, termed floral developmental homeostasis.