Dr Charlotte Kirchhelle
From edge to organ: the role of cell geometry in plant morphogenesis.
How do organisms develop the diverse anatomical shapes observed in nature? Answering this question is pivotal for our basic understanding of multicellular organisms and the rational improvement of domesticated species. However, the underlying process of morphogenesis is highly complex: it involves integrating different kinds of information (genetic, biochemical, biomechanical, and geometric) across multiple scales in space and time. Since this complexity poses a significant challenge to traditional experimental approaches, I will adopt an interdisciplinary approach combining classic molecular biology with state-of-the-art quantitative imaging and computational modelling to study plant morphogenesis, focussing on the recently identified role of cell geometric edges.
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The importance of being edgy: cell geometric edges as an emerging polar domain in plant cells.
June 2020|Journal article|Journal of microscopyPolarity is an essential feature of multicellular organisms and underpins growth and development as well as physiological functions. In polyhedral plant cells, polar domains at different faces have been studied in detail. In recent years, cell edges (where two faces meet) have emerged as discrete spatial domains with distinct biochemical identities. Here, we review and discuss recent advances in our understanding of cell edges as functional polar domains in plant cells and other organisms, highlighting conceptual parallels and open questions regarding edge polarity. -
Optimizing Rhizobium-legume symbioses by simultaneous measurement of rhizobial competitiveness and N2 fixation in nodules.
May 2020|Journal article|Proceedings of the National Academy of Sciences of the United States of AmericaLegumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N<sub>2</sub>) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N<sub>2</sub> fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N<sub>2</sub> fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different <i>Rhizobium leguminosarum</i> strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple <i>Rhizobium</i>-legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.Rhizobium leguminosarum, Fabaceae, Peas, Nitrogen, Green Fluorescent Proteins, Soil Microbiology, Nitrogen Fixation, Symbiosis, Plasmids, Root Nodules, Plant, Synthetic Biology, High-Throughput Nucleotide Sequencing -
Spatio-temporal control of post-Golgi exocytic trafficking in plants.
February 2020|Journal article|Journal of cell scienceA complex and dynamic endomembrane system is a hallmark of eukaryotic cells and underpins the evolution of specialised cell types in multicellular organisms. Endomembrane system function critically depends on the ability of the cell to (1) define compartment and pathway identity, and (2) organise compartments and pathways dynamically in space and time. Eukaryotes possess a complex molecular machinery to control these processes, including small GTPases and their regulators, SNAREs, tethering factors, motor proteins, and cytoskeletal elements. Whereas many of the core components of the eukaryotic endomembrane system are broadly conserved, there have been substantial diversifications within different lineages, possibly reflecting lineage-specific requirements of endomembrane trafficking. This Review focusses on the spatio-temporal regulation of post-Golgi exocytic transport in plants. It highlights recent advances in our understanding of the elaborate network of pathways transporting different cargoes to different domains of the cell surface, and the molecular machinery underpinning them (with a focus on Rab GTPases, their interactors and the cytoskeleton). We primarily focus on transport in the context of growth, but also highlight how these pathways are co-opted during plant immunity responses and at the plant-pathogen interface. -
The Formation of a Camalexin Biosynthetic Metabolon.
November 2019|Journal article|The Plant cellArabidopsis (<i>Arabidopsis thaliana</i>) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in <i>GSTU4</i>-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism.Plants, Genetically Modified, Arabidopsis, Tobacco, Plant Leaves, Sesquiterpenes, Thiazoles, Indoles, Cytochrome P-450 Enzyme System, Glutathione Transferase, Arabidopsis Proteins, Gene Expression Regulation, Plant, Biosynthetic Pathways, Gene Knockout Techniques -
Two mechanisms regulate directional cell growth in Arabidopsis lateral roots.
July 2019|Journal article|eLifeMorphogenesis in plants depends critically on directional (anisotropic) growth. This occurs principally perpendicular to the net orientation of cellulose microfibrils (CMFs), which is in turn controlled by cortical microtubules (CMTs). In young lateral roots of <i>Arabidopsis thaliana</i>, growth anisotropy also depends on RAB-A5c, a plant-specific small GTPase that specifies a membrane trafficking pathway to the geometric edges of cells. Here we investigate the functional relationship between structural anisotropy at faces and RAB-A5c activity at edges during lateral root development. We show that surprisingly, inhibition of RAB-A5c function is associated with increased CMT/CMF anisotropy. We present genetic, pharmacological, and modelling evidence that this increase in CMT/CMF anisotropy partially compensates for loss of an independent RAB-A5c-mediated mechanism that maintains anisotropic growth in meristematic cells. We show that RAB-A5c associates with CMTs at cell edges, indicating that CMTs act as an integration point for both mechanisms controlling cellular growth anisotropy in lateral roots.Microtubules, Arabidopsis, Plant Roots, rab GTP-Binding Proteins, Arabidopsis Proteins, Cell Proliferation, Morphogenesis, Plant Cells -
Universal Methods for Transgene Induction Using the Dexamethasone-Inducible Transcription Activation System pOp6/LhGR in Arabidopsis and Other Plant Species.
March 2019|Journal article|Current protocols in plant biologyUse of chemically inducible systems for transgene expression is a crucial requirement for modern plant biology research, as it allows (1) expression of transgenes that compromise plant viability or fertility when constitutively expressed and (2) spatiotemporal control of transgene expression levels. We describe the stringently regulated and highly responsive dexamethasone-inducible gene expression system pOp6/LhGR, which comprises the chimeric transcription activator LhGR and the corresponding pOp6 promoter. Upon induction, the LhGR activator binds to the pOp6 promoter and induces expression of the target gene of interest. We provide detailed protocols for inducing transgene expression at different developmental stages and in different plant species and discuss dexamethasone stability and use of its analogs. We also introduce new, versatile, GATEWAY-compatible binary vectors that are now available for the pOp6/LhGR system. © 2019 by John Wiley & Sons, Inc. -
A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development.
May 2017|Journal article|Journal of visualized experiments : JoVESeveral aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.Arabidopsis, Hypocotyl, Plant Roots, Microscopy, Confocal -
The Specification of Geometric Edges by a Plant Rab GTPase Is an Essential Cell-Patterning Principle During Organogenesis in Arabidopsis.
February 2016|Journal article|Developmental cellPlant organogenesis requires control over division planes and anisotropic cell wall growth, which each require spatial patterning of cells. Polyhedral plant cells can display complex patterning in which individual faces are established as biochemically distinct domains by endomembrane trafficking. We now show that, during organogenesis, the Arabidopsis endomembrane system specifies an important additional cellular spatial domain: the geometric edges. Previously unidentified membrane vesicles lying immediately beneath the plasma membrane at cell edges were revealed through localization of RAB-A5c, a plant GTPase of the Rab family of membrane-trafficking regulators. Specific inhibition of RAB-A5c activity grossly perturbed cell geometry in developing lateral organs by interfering independently with growth anisotropy and cytokinesis without disrupting default membrane trafficking. The initial loss of normal cell geometry can be explained by a failure to maintain wall stiffness specifically at geometric edges. RAB-A5c thus meets a requirement to specify this cellular spatial domain during organogenesis.Cell Membrane, Arabidopsis, rab GTP-Binding Proteins, Cytokinesis, Protein Transport, Organogenesis, Plant Cells