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The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.
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Embriófitas , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/metabolismo , Embriófitas/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Fosforilação , Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Algas/metabolismoRESUMO
Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploited a real-time salt-induced tilting assay. This assay follows root growth upon both gravitropic and salt challenges, revealing that root bending upon tilting is modulated by Na+ ions, but not by osmotic stress. Next, we measured this salt-specific response in 345 natural Arabidopsis (Arabidopsis thaliana) accessions and discovered a genetic locus, encoding the cell wall-modifying enzyme EXTENSIN ARABINOSE DEFICIENT TRANSFERASE (ExAD) that is associated with root bending in the presence of NaCl (hereafter salt). Extensins are a class of structural cell wall glycoproteins known as hydroxyproline (Hyp)-rich glycoproteins, which are posttranslationally modified by O-glycosylation, mostly involving Hyp-arabinosylation. We show that salt-induced ExAD-dependent Hyp-arabinosylation influences root bending responses and cell wall thickness. Roots of exad1 mutant seedlings, which lack Hyp-arabinosylation of extensin, displayed increased thickness of root epidermal cell walls and greater cell wall porosity. They also showed altered gravitropic root bending in salt conditions and a reduced salt-avoidance response. Our results suggest that extensin modification via Hyp-arabinosylation is a unique salt-specific cellular process required for the directional response of roots exposed to salinity.
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Plant external surfaces are often covered by barriers that control the exchange of molecules, protect from pathogens and offer mechanical integrity. A key question is when and how such surface barriers are generated. Post-embryonic surfaces have well-studied barriers, including the cuticle, and it has been previously shown that the late Arabidopsis thaliana embryo is protected by an endosperm-derived sheath deposited onto a primordial cuticle. Here, we show that both cuticle and sheath are preceded by another structure during the earliest stages of embryogenesis. This structure, which we named the embryonic envelope, is tightly wrapped around the embryonic surface but can be physically detached by cell wall digestion. We show that this structure is composed primarily of extensin and arabinogalactan O-glycoproteins and lipids, which appear to form a dense and elastic crosslinked embryonic envelope. The envelope forms in cuticle-deficient mutants and in a mutant that lacks endosperm. This embryo-derived envelope is therefore distinct from previously described cuticle and sheath structures. We propose that it acts as an expandable diffusion barrier, as well as a means to mechanically confine the embryo to maintain its tensegrity during early embryogenesis.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sementes/genética , Endosperma/genética , Difusão , Regulação da Expressão Gênica de PlantasRESUMO
The devastating pathogen Botrytis cinerea infects a broad spectrum of host plants, causing great socio-economic losses. The necrotrophic fungus rapidly kills plant cells, nourishing their wall and cellular contents. To this end, necrotrophs secrete a cocktail of cell wall degrading enzymes, phytotoxic proteins and metabolites. Additionally, many fungi produce specialized invasion organs that generate high invasive pressures to force their way into the plant cell. However, for most necrotrophs, including Botrytis, the biomechanics of penetration and its contribution to virulence are poorly understood. Here, we use a combination of quantitative micromechanical imaging and CRISPR-Cas-guided mutagenesis to show that Botrytis uses substantial invasive pressure, in combination with strong surface adherence, for penetration. We found that the fungus establishes a unique mechanical geometry of penetration that develops over time during penetration events, and which is actin cytoskeleton dependent. Furthermore, interference of force generation by blocking actin polymerization was found to decrease Botrytis virulence, indicating that also for necrotrophs, mechanical pressure is important in host colonization. Our results demonstrate for the first time mechanistically how a necrotrophic fungus such as Botrytis employs this 'brute force' approach, in addition to the secretion of lytic proteins and phytotoxic metabolites, to overcome plant host resistance.
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Inkjet printing is a ubiquitous consumer and industrial process that involves concomitant processes of droplet impact, wetting, evaporation, and imbibement into a substrate as well as consequential substrate rearrangements and remodeling. In this work, we perform a study on the interaction between ink dispersions of different composition on substrates of increasing complexity to disentangle the motion of the liquid from the dynamic response of the substrate. We print three variations of pigmented inks and follow the ensuing dynamics at millisecond and micron time and length scales until complete drying using a multiple scattering technique, laser speckle imaging (LSI). Measurements of the photon transport mean free path, l*, for the printed inks and substrates show that the spatial region of information capture is the entire droplet volume and a depth within the substrate of a few µm beneath the droplet. Within this spatial confinement, LSI is an ideal approach for studying the solid-liquid transition at these small length and time scales by obtaining valid g2 and d2 autocorrelation functions and interpreting these dynamic changes under through kymographs. Our in situ LSI results show that droplets undergo delamination and cracking processes arising during droplet drying, which are confirmed by post mortem SEM imaging.
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The drying of multi-component dispersions is a common phenomenon in a variety of everyday applications, including coatings, inks, processed foods, and cosmetics. As the solvent evaporates, the different components may spontaneously segregate laterally and/or in depth, which can significantly impact the macroscopic properties of the dried film. To obtain a quantitative understanding of these processes, high-resolution analysis of segregation patterns is crucial. Yet, current state-of-the-art methods are limited to transparent, non-deformable labeled colloids, limiting their applicability. In this study, we employ three techniques that do not require customized samples, as their imaging contrast relies on intrinsic variations in the chemical nature of the constituent species: confocal Raman microscopy, cross-sectional Raman microscopy, and a combination of scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDX). For broad accessibility, we offer a thorough guide to our experimental steps and data analysis methods. We benchmark the capabilities on a film that dries homogeneously at room temperature but exhibits distinct segregation features at elevated temperature, notably self-stratification, i.e., autonomous layer formation, due to a colloidal size mismatch. Confocal Raman microscopy offers a direct means to visualize structures in three dimensions without pre-treatment, its accuracy diminishes deeper within the film, making cross-sectional Raman imaging and SEM-EDX better options. The latter is the most elaborate method, yet we show that it can reveal the most subtle and small-scale microseparation of the two components in the lateral direction. This comparative study assists researchers in choosing and applying the most suitable technique to quantify structure formation in dried multi-component films.
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Mechanical patterns control a variety of biological processes in plants. The microviscosity of cellular structures effects the diffusion rate of molecules and organelles, thereby affecting processes such as metabolism and signaling. Spatial variations in local viscosity are also generated during fundamental events in the cell life cycle. While crucial to a complete understanding of plant mechanobiology, resolving subcellular microviscosity patterns in plants has remained an unsolved challenge. We present an imaging microviscosimetry toolbox of molecular rotors that yield complete microviscosity maps of cells and tissues, specifically targeting the cytosol, vacuole, plasma membrane, and wall of plant cells. These boron-dipyrromethene (BODIPY)-based molecular rotors are rigidochromic by means of coupling the rate of an intramolecular rotation, which depends on the mechanics of their direct surroundings, with their fluorescence lifetime. This enables the optical mapping of fluidity and porosity patterns in targeted cellular compartments. We show how apparent viscosity relates to cell function in the root, how the growth of cellular protrusions induces local tension, and how the cell wall is adapted to perform actuation surrounding leaf pores. These results pave the way to the noninvasive micromechanical mapping of complex tissues.
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Modelos Biológicos , Células Vegetais , Fenômenos Fisiológicos Vegetais , Viscosidade , Corantes Fluorescentes/química , Proteínas Motores Moleculares/metabolismo , Sondas Moleculares/química , Especificidade de Órgãos , Organelas/metabolismoRESUMO
Single stranded DNA (ssDNA), or another polyanion, can be mixed with polycations to form liquid-like complex coacervates. When the polycations are replaced by cationic-neutral diblock copolymers, complex coacervate core micelles (C3Ms) can be formed instead. In both complex coacervates and C3Ms, dynamics plays an important role. Yet, to date, the effect of chain length on the dynamics effect is still not fully understood. The DNA complexes provide a versatile platform to further elucidate these chain length effects because the DNA is monodisperse and its length can be easily adapted. Therefore, we study in this paper the dynamics of fluorescently labelled ssDNA in both complex coacervate droplets and micelles. The DNA dynamics in the complex coacervate droplets is probed by fluorescence recovery after photobleaching (FRAP). We observe that the DNA diffusion coefficient depends more strongly on the DNA length than predicted by the sticky Rouse model and we show that this can be partly explained by changes in complex coacervate density, but that also other factors might play a role. We measure the molecular exchange of C3Ms by making use of Förster resonance energy transfer (FRET) and complement these measurements with Langevin dynamics simulations. We conclude that chain length polydispersity is the main cause of a broad distribution of exchange rates. We hypothesise that the different exchange rates that we observe for the monodisperse DNA are mainly caused by differences in dye interactions and show that the dye can indeed have a large effect on the C3M exchange. In addition, we show that a new description of the C3M molecular exchange is required that accounts among others for the effect of the length of the oppositely charged core species. Together our findings can help to better understand the dynamics in both specific DNA systems and in complex coacervate droplets and micelles in general.
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Micelas , Polímeros , Cátions , DNA , Transferência Ressonante de Energia de FluorescênciaRESUMO
The application of complex coacervates in promising areas such as coatings and surgical glues requires a tight control of their viscous and elastic behaviour, and a keen understanding of the corresponding microscopic mechanisms. While the viscous, or dissipative, aspect is crucial at pre-setting times and in preventing detachment, elasticity at long waiting times and low strain rates is crucial to sustain a load-bearing joints. The independent tailoring of dissipative and elastic properties proves to be a major challenge that can not be addressed adequately by the complex coacervate motif by itself. We propose a versatile model of complex coacervates with customizable rheological fates by functionalization of polyelectrolytes with terpyridines, which provide transient crosslinks through complexation with metals. We show that the rheology of the hybrid complexes shows distinct footprints of both metal-ligand and coacervate dynamics, the former as a contribution very close to pure Maxwell viscoelasticity, the latter approaching a sticky Rouse fluid. Strikingly, when the contribution of metal-ligand bonds is dominant at long times, the relaxation of the overall complex is much slower than either the "native" coacervate relaxation time or the dissociation time of a comparable non-coacervate polyelectrolyte-metal-ligand complex. We recognize this slowing-down of transient bonds as a synergistic effect that has important implications for the use of complementary transient bonding in coacervate complexes.
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In the study of colloidal glasses, crystallization is often suppressed by leveraging size polydispersity, ranging from systems where particle sizes exhibit a continuous distribution to systems composed of particles of two or more distinct sizes. The effects of the disparities in size of the particles on the colloidal glass transition are not yet completely understood. Especially, the question of the existence of a decoupled glass transition between the large and small population remains. In order to measure colloidal dynamics on very long time scales and to disentangle the dynamics of the two populations, we employ contrast variation multispeckle diffusing wave spectroscopy. With this method, we aim to analyze the effect of size ratio, a = rPS/ rpNIPAM, on particle dynamics near the glass transition of a binary colloidal system. We find that both for long-time (α-) and short-time (ß-) relaxation, the dynamics of the small particles either completely decouple from the large ones ( a = 0.2), moving freely through a glassy matrix, or are identical to the dynamics of the larger-sized population ( a = 0.37 and 1.44). For a size ratio of 0.37, we find a single-glass transition for both particle populations. The postulated double-glass transition in simulations and theory is not observed.
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The formation of non-hexagonal crystalline structures by the organisation of colloidal nanoparticles often involves the use of complex particles with anisotropic shape or interactions or the imposition of non-uniform external fields. Here we explore how unusual symmetries can be created using experimentally realistic particles that interact through isotropic and purely repulsive potentials. In particular, we use simulations to explore the phase behavior of two-dimensional systems of star polymers. We uncover how the tail of the pair potential has a large role in dictating the phase behavior. Star polymers interacting in the far field with a Gaussian potential only form hexagonal phases, while an exponential tail gives rise to stable primitive oblique and honeycomb lattices. We identify the ratio in strength between long and short range interactions and the nature of the transition between these regimes as crucial parameters to predict when non-hexagonal crystals of star polymers can be stable. This leads to experimental design rules for creating star polymers which should exhibit unusual lattice formation.
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Colloidal gels are space-spanning networks of aggregated particles. The mechanical response of colloidal gels is governed, to a large extent, by the properties of the individual gel strands. To study how colloidal gels respond to repeated deformations, we perform Brownian dynamics simulations on single strands of aggregated colloidal particles. While current models assume that gel failure is due to the brittle rupture of gel strands, our simulations show that gel strands undergo large plastic deformations prior to breaking. Rearrangement of particles within the strands leads to plastic lengthening and softening of the strands, which may ultimately lead to strand necking and ductile failure. This failure mechanism occurs irrespective of the thickness and length of the strands and the range and strength of the interaction potential. Rupture of gel strands is more likely for long and thin strands and for a long-ranged interaction potential.
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The dynamics of interstitial dopants govern the properties of a wide variety of doped crystalline materials. To describe the hopping dynamics of such interstitial impurities, classical approaches often assume that dopant particles do not interact and travel through a static potential energy landscape. Here we show, using computer simulations, how these assumptions and the resulting predictions from classical Eyring-type theories break down in entropically stabilized body-centered cubic (BCC) crystals due to the thermal excitations of the crystalline matrix. Deviations are particularly severe close to melting where the lattice becomes weak and dopant dynamics exhibit strongly localized and heterogeneous dynamics. We attribute these anomalies to the failure of both assumptions underlying the classical description: (i) The instantaneous potential field experienced by dopants becomes largely disordered due to thermal fluctuations and (ii) elastic interactions cause strong dopant-dopant interactions even at low doping fractions. These results illustrate how describing nonclassical dopant dynamics requires taking the effective disordered potential energy landscape of strongly excited crystals and dopant-dopant interactions into account.
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The repeated loading of a solid leads to microstructural damage that ultimately results in catastrophic material failure. While posing a major threat to the stability of virtually all materials, the microscopic origins of fatigue, especially for soft solids, remain elusive. Here we explore fatigue in colloidal gels as prototypical inhomogeneous soft solids by combining experiments and computer simulations. Our results reveal how mechanical loading leads to irreversible strand stretching, which builds slack into the network that softens the solid at small strains and causes strain hardening at larger deformations. We thus find that microscopic plasticity governs fatigue at much larger scales. This gives rise to a new picture of fatigue in soft thermal solids and calls for new theoretical descriptions of soft gel mechanics in which local plasticity is taken into account.
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A plethora of natural systems rely on the consumption of chemical fuel or input of external energy to control the assembly and disassembly of functional structures on demand. While dissipative assembly has been demonstrated, the control of structural breakdown using a dissipative cycle remains almost unexplored. Here, we propose and realize a dissipative disassembly process using two coupled cyclic reactions, in which protons mediate the interaction between the cycles. We show how an ordered colloidal crystal, can cyclically transform into a disordered state by addition of energy to a chemical cycle, reversibly activating a photoacid. This cycle is coupled to the colloidal assembly cycle via the exchange of protons, which in turn trigger charging of the particles. This system is an experimental realization of a cyclic reaction-assembly network and its principle can be extended to other types of structure formation.
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The quest to unravel the nature of the glass transition, where the viscosity of a liquid increases by many orders of magnitude, while its static structure remains largely unaffected, remains unresolved. While various structural and dynamical precursors to vitrification have been identified, a predictive and quantitative description of how subtle changes at the microscopic scale give rise to the steep growth in macroscopic viscosity is missing. It was recently proposed that the presence of long-lived bonded structures within the liquid may provide the long-sought connection between local structure and global dynamics. Here we directly observe and quantify the connectivity dynamics in liquids of charged colloids en route to vitrification using three-dimensional confocal microscopy. We determine the dynamic structure from the real-space van Hove correlation function and from the particle trajectories, providing upper and lower bounds on connectivity dynamics. Based on these data, we extend Dyre's model for the glass transition to account for particle-level structural dynamics; this results in a microscopic expression for the slowing down of relaxations in the liquid that is in quantitative agreement with our experiments. These results indicate how vitrification may be understood as a dynamical connectivity transition with features that are strongly reminiscent of rigidity percolation scenarios.
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Here, a means of controlling the assembly pathways of p-conjugated oligoelectrolytes into supramolecular fibers and microtubes is presented, and it is shown how the addition of small end-caps to well-defined and pH-responsive conjugated oligomers can alter the balance between repulsive and attractive supramolecular forces and enables control of the morphology of the hierarchical assembly process. The assembly stages from nuclei to protofibers are evidenced and a hypothesis on the mechanism of microtubes formation using a combination of analytical methods is provided, revealing different degrees of order at different scales along the structural hierarchy.
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The coassembly of well-defined biological nanostructures relies on a delicate balance between attractive and repulsive interactions between biomolecular building blocks. Viral capsids are a prototypical example, where coat proteins exhibit not only self-interactions but also interact with the cargo they encapsulate. In nature, the balance between antagonistic and synergistic interactions has evolved to avoid kinetic trapping and polymorphism. To date, it has remained a major challenge to experimentally disentangle the complex kinetic reaction pathways that underlie successful coassembly of biomolecular building blocks in a noninvasive approach with high temporal resolution. Here we show how macromolecular force sensors, acting as a genome proxy, allow us to probe the pathways through which a viromimetic protein forms capsids. We uncover the complex multistage process of capsid assembly, which involves recruitment and complexation, followed by allosteric growth of the proteinaceous coat. Under certain conditions, the single-genome particles condense into capsids containing multiple copies of the template. Finally, we derive a theoretical model that quantitatively describes the kinetics of recruitment and growth. These results shed new light on the origins of the pathway complexity in biomolecular coassembly.
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Fluorescent nanodiamonds are gaining increasing attention as fluorescent labels in biology in view of the fact that they are essentially nontoxic, do not bleach, and can be used as nanoscale sensors for various physical and chemical properties. To fully realize the nanosensing potential of nanodiamonds in biological applications, two problems need to be addressed: their limited colloidal stability, especially in the presence of salts, and their limited ability to be taken up by cells. We show that the physical adsorption of a suitably designed recombinant polypeptide can address both the colloidal stability problem and the problem of the limited uptake of nanodiamonds by cells in a very straightforward way, while preserving both their spectroscopic properties and their excellent biocompatibility.
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Coloides/química , Nanodiamantes/química , Proteínas Recombinantes/química , Adsorção , Transporte Biológico , Linhagem Celular Tumoral , Coloides/farmacocinética , Coloides/toxicidade , Fluorescência , Humanos , Luz , Nanodiamantes/efeitos da radiação , Nanodiamantes/toxicidade , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidadeRESUMO
Crystals with low latent heat are predicted to melt from an entropically stabilized body-centered cubic symmetry. At this weakly first-order transition, strongly correlated fluctuations are expected to emerge, which could change the nature of the transition. Here we show how large fluctuations stabilize bcc crystals formed from charged colloids, giving rise to strongly power-law correlated heterogeneous dynamics. Moreover, we find that significant nonaffine particle displacements lead to a vanishing of the nonaffine shear modulus at the transition. We interpret these observations by reformulating the Born-Huang theory to account for nonaffinity, illustrating a scenario of ordered solids reaching a state where classical lattice dynamics fail.