In plants, polar cell growth is essential for processes such as root hair and pollen tube growth, but how it integrates with nuclear movement, cytoskeletal organization, and cell mechanics is not fully understood. We conducted high-resolution live imaging of Arabidopsis thaliana root hairs throughout their development using a microfluidic device. We identified 3 distinct stages--fast growth, slow growth, and early maturation--and quantified growth kinetics at high temporal resolution. The transition from fast to slow growth was consistent with cytoskeletal dynamics causing reduced tip growth and decreased nucleus-tip distance. Based on these observations, we developed a mathematical model linking cytoskeletal dynamics with tip growth and nuclear dynamics. Through genetic and pharmacological approaches, we were able to disrupt or trigger this transition, supporting the model and revealing the existence of essential crosstalk between actin filaments and microtubules. Additionally, vacuole dynamics, root hair diameter, and cell stiffness changed during the fast-to-slow transition, indicating a coordinated regulation of multiple subcellular systems. Together, these results connect nuclear, cytoskeletal, and mechanical dynamics during root hair development, offering an integrated view of the subcellular processes behind the control of polar cell growth in plants.

Synthetic insecticides are recognized as a major factor contributing to the global decline in insect abundance and diversity. Within this context, anthranilic diamides have gained increasing market share in Europe since 2018. We investigated the second-generation diamide cyantraniliprole, which induces aberrant cytoplasmic calcium release in honeybee skeletal muscle cells. In vivo assays demonstrated that acute contact toxicity varied with exposure site, being more severe when applied to the abdomen, antennae, or ventral thorax compared with the dorsal thorax. Following thoracic exposure to sublethal doses, continuous 21-h monitoring revealed a dose-dependent alteration of behavior, with a reduction in locomotor activity, including maximal velocity and total distance traveled. Notably, at the lowest concentration, antennal exposure elicited stronger behavioral impairments, indicating potential disruption of sensory processing and environmental cue perception. Importantly, cyantraniliprole and its parent compound, chlorantraniliprole, also triggered anarchic calcium mobilization in mammalian skeletal muscle fibers, highlighting potential cross-taxa effects. These results strengthen previous evidence of diamides toxicity in bee cardiomyocytes and neurons and emphasize significant gaps in current risk assessment frameworks. Collectively, our findings indicate that anthranilic diamides represent not only an ecological threat to pollinators but also raise concerns for mammalian health, warranting more stringent evaluation of this insecticide class.

Synthetic insecticides used in agriculture are one of the major causes for the loss of biodiversity. Naturally-occurring substances have been envisioned by agrochemical companies as environmentally-friendly alternatives. Here, the effects of the plant alkaloid veratridine and a synthetic pyrethroid insecticide, lambda-cyhalothrin, were compared on honey bees, using electrophysiology, ethology, and classical toxicology. Both compounds triggered strong deleterious effects, in vitro and in vivo. In cell-cultured antennal lobe neurons, their effects were qualitatively similar with the appearance of a persistent tail current through voltage-gated sodium channels. Veratridine-induced tail current had much slower kinetics. Both compounds caused mortality in a similar range of doses. At sublethal exposure levels, only lambda-cyhalothrin caused strong behavioral defects. This work could pave the way to updating the current safety assessment framework for insecticides, showing that in vitro assays can help in anticipating lethal and sublethal toxicity of both natural and synthetic compounds.

We describe a new software library written in the Python programming language to perform computational imaging on the so-called EnderScope, an automated imaging system based on the 3-axis motion stage of a 3D printer. This library is designed to be easy to use for beginner users and provides high-level functions to access used devices. We provide practical use cases and example codes using the library as an educational tool for testing, experimenting or developing computational imaging and smart microscopy algorithms.

Polar cell growth is a fundamental process across organisms, yet its coordination with nuclear movement and cytoskeleton dynamics remains underexplored. Focusing on Arabidopsis thaliana root hairs, we investigate these processes using high-resolution live imaging within microfluidics-based experiments. By incorporating data on cytoskeletal dynamics, nuclear positioning, and tip growth into a mathematical model, we analyse how their interactions shape the different growth phases that we reveal for the first time in this study. Chemical treatments and mutant analyses further support our model, revealing that timely cytoskeletal changes drive transitions between these growth phases, and correlate with shifts in nuclear movement and morphology. This regulation suggests a microtubule-actin crosstalk in the root hair subapical region. Additionally, we present novel findings on vacuole movement and cell stiffness, further refining our understanding of tip growth dynamics. Collectively, our work provides a comprehensive framework for understanding how transitions between growth phases are orchestrated in plant tip-growing cells. ### Competing Interest Statement The authors have declared no competing interest.

Streptomycetaceae are found ubiquitously within plant microbiota. Several species belonging to this family are plant growth-promoting bacteria or may inhibit phytopathogens. Such bacteria therefore exert crucial functions in host development and resistance to stresses. Recent studies have shown that plants select beneficial bacteria into their microbiota. However, the selection process and the molecular mechanisms by which selected bacteria modulate the physiology of their host are not yet fully understood. Previous work revealed that the metabolic status of Arabidopsis thaliana was crucial for the selection of Streptomycetaceae into the microbiota, in particular bacteria phylogenetically related to Streptomyces cocklensis or Actinacidiphila bryophytorum (previously named Streptomyces bryophytorum). Here, the Arabidopsis-Streptomycetaceae interaction was further depicted by inoculating axenic A. thaliana with S. cocklensis DSM 42063 or A. bryophytorum DSM 42138. We showed that these two bacteria colonize A. thaliana ecotype Columbia-0 plants, but the colonization efficiency is reduced in a chs5 mutant of the same ecotype, being altered in isoprenoid, phenylpropanoid, and lipid profiles. We observed that A. bryophytorum inhibits growth of the chs5 mutant but not of the wild type, suggesting that the Arabidopsis-Actinacidiphila interaction depends on the metabolic status of the host. Using a mass spectrometry-based proteomic approach, we showed that S. cocklensis and A. bryophytorum modulate the A. thaliana proteome, in particular components involved in photosynthesis or phytohormone homeostasis. This study unveils specific aspects of the Arabidopsis-Streptomycetaceae interaction and highlights its complexity and diversity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

AbstractIn honey bees, circulation of blood (hemolymph) is driven by the peristaltic contraction of the heart vessel located in the dorsal part of the abdomen. Chlorantraniliprole (CHL) is an insecticide of the anthranilic diamide class which main mode of action is to alter the function of intracellular Ca2+release channels (known as RyRs, for ryanodine receptors). In the honey bee, it was recently found to be more toxic when applied on the dorsal part of the abdomen, suggesting a direct cardiotoxicity. In the present study, a short-term exposure of semi-isolated bee hearts to CHL (0.1–10 µM) induces alterations of cardiac contraction. These alterations range from a slow-down of systole and diastole kinetics, to bradycardia and cardiac arrest. The bees heart wall is made of a single layer of semi-circular cardiomyocytes arranged concentrically all along the long axis of tube lumen. Since the heart tube is suspended to the cuticle through long tubular muscles fibers (so-called alary muscle cells), the CHL effects inex-vivoheart preparations could result from the modulation of RyRs present in these skeletal muscle fibers as well as cardiomyocytes RyRs themselves. In order to specifically assess effects of CHL on cardiomyocytes, for the first time, intact heart cells were enzymatically dissociated from bees. Exposure of cardiomyocytes to CHL induces an increase in cytoplasmic calcium, cell contraction at the highest concentrations and depletion of intracellular stores. Electrophysiological properties of isolated cardiomyocytes were described, with a focus on voltage-gated Ca2+channels responsible for the cardiac action potentials depolarization phase. Two types of Ca2+currents were measured under voltage-clamp. Exposure to CHL was accompanied by a decrease in voltage-activated Ca2+currents densities. Altogether, these results show that chlorantraniliprole can cause cardiac defects in honey bees.

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signalling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this novel degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signalling activity in the SAM. We show that high GA signalling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organisation of internodes, thus contributing to internode specification in the SAM.

The current risk assessment framework for insecticides suffers from certain shortcomings in adequately addressing the effects of low doses on off-target species. To remedy this gap, a combination of behavioural assays and in vitro cellular approaches are required to refine the precision of toxicity assessment. The domestic honey bee has long been standing as an emblematic pollinator in ecotoxicology, and once more, it provides us with a practical testing model for this purpose. First, newly emerged bees (D1) were found more vulnerable than 6 days-old bees (D6) to deltamethrin, a widely used α-cyano-3-phenoxybenzyle pyrethroid. In D1 bees, the range of doses inducing mortality was shifted towards lower values (∼2-fold) with a correspondingly lower LD50 (11 ng/bee). Moreover, at low doses that do not induce mortality in laboratory conditions, the locomotor behaviour of D1 bees was more impacted than in D6 bees. This was evidenced by an increase in immobility time and a decrease in locomotor performance across all tested doses for D1 bees (0.75, 1.5 and 3 ng/bee) during automated 21 h-long observations. Behavioural disorders are linked to deltamethrin's disruption of voltage-gated sodium channels (NaVs) functions, as quantified in cultured neuronal cells. In the presence of deltamethrin, patch-clamp experiments revealed a concentration- and a use-dependent slowing of NaV kinetics. Channel's deactivation is slowed by three orders of magnitude at 10 μM deltamethrin. Two additional phenoxybenzyle pyrethroids, including the commonly used cypermethrin, elicited quantitatively similar effects on NaV kinetics. The integration of in vitro cellular assays and behavioural assays may facilitate a deeper understanding and prediction of insecticides toxicity.

Low-cost and scalable technologies that allow people to measure microplastics in their local environment could facilitate a greater understanding of the global problem of marine microplastic pollution. A typical way to measure marine microplastic pollution involves imaging filtered seawater samples stained with a fluorescent dye to aid in the detection of microplastics. Although traditional fluorescence microscopy allows these particles to be manually counted and detected, this is a resource- and labour-intensive task. Here, we describe a novel, low-cost microscope for automated scanning and detection of microplastics in filtered seawater samples-the EnderScope. This microscope is based on the mechanics of a low-cost 3D printer (Creality Ender 3). The hotend of the printer is replaced with an optics module, allowing for the reliable and calibrated motion system of the 3D printer to be used for automated scanning over a large area (>20 × 20 cm). The EnderScope is capable of both reflected light and fluorescence imaging. In both configurations, we aimed to make the design as simple and cost-effective as possible, for example, by using low-cost LEDs for illumination and lighting gels as emission filters. We believe this tool is a cost-effective solution for microplastic measurement. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

Emerging evidence indicates that in addition to its well-recognized functions in antiviral RNA silencing, dsRNA elicits pattern-triggered immunity (PTI), likely contributing to plant resistance against virus infections. However, compared to bacterial and fungal elicitor-mediated PTI, the mode-of-action and signaling pathway of dsRNA-induced defense remain poorly characterized. Here, using multicolor in vivo imaging, analysis of GFP mobility, callose staining, and plasmodesmal marker lines in Arabidopsis thaliana and Nicotiana benthamiana, we show that dsRNA-induced PTI restricts the progression of virus infection by triggering callose deposition at plasmodesmata, thereby likely limiting the macromolecular transport through these cell-to-cell communication channels. The plasma membrane-resident SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1, the BOTRYTIS INDUCED KINASE1/AVRPPHB SUSCEPTIBLE1-LIKE KINASE1 kinase module, PLASMODESMATA-LOCATED PROTEINs 1/2/3, as well as CALMODULIN-LIKE 41 and Ca2+ signals are involved in the dsRNA-induced signaling leading to callose deposition at plasmodesmata and antiviral defense. Unlike the classical bacterial elicitor flagellin, dsRNA does not trigger a detectable reactive oxygen species (ROS) burst, substantiating the idea that different microbial patterns trigger partially shared immune signaling frameworks with distinct features. Likely as a counter strategy, viral movement proteins from different viruses suppress the dsRNA-induced host response leading to callose deposition to achieve infection. Thus, our data support a model in which plant immune signaling constrains virus movement by inducing callose deposition at plasmodesmata and reveals how viruses counteract this layer of immunity.

Biosolids are byproducts of wastewater treatment. With the increasing global population, the amounts of wastewater to be treated are expanding, along with the amounts of biosolids generated. The reuse of biosolids is now accepted for diversified applications in fields such as agriculture, engineering, agro-forestry. However, biosolids are known to be potential carriers of compounds that can be toxic to living beings or alter the environment. Therefore, biosolid reuse is subject to regulations, mandatory analyses are performed on heavy metals, persistent organic pollutants or pathogens. Conventional methods for the analysis of heavy metals and persistent organic pollutants are demanding, lengthy, and sometimes unsafe. Here, we propose mass spectrometry imaging as a faster and safer method using small amounts of material to monitor heavy metals and persistent organic pollutants in different types of biosolids, allowing for ecological and health risk assessment before reuse. Our methodology can be extended to other soil-like matrices.

The combination of ever-increasing microscopy resolution with cytogenetical tools allows for detailed analyses of nuclear functional partitioning. However, the need for reliable qualitative and quantitative methodologies to detect and interpret chromatin sub-nuclear organization dynamics is crucial to decipher the underlying molecular processes. Having access to properly automated tools for accurate and fast recognition of complex nuclear structures remains an important issue. Cognitive biases associated with human-based curation or decisions for object segmentation tend to introduce variability and noise into image analysis. Here, we report the development of two complementary segmentation methods, one semi-automated (iCRAQ) and one based on deep learning (Nucl.Eye.D), and their evaluation using a collection of A. thaliana nuclei with contrasted or poorly defined chromatin compartmentalization. Both methods allow for fast, robust and sensitive detection as well as for quantification of subtle nucleus features. Based on these developments, we highlight advantages of semi-automated and deep learning-based analyses applied to plant cytogenetics.

The deposition and turnover of callose (beta-1,3 glucan polymer) in the cell wall surrounding the neck regions of plasmodesmata (PD) controls the cell-to-cell diffusion rate of molecules and, therefore, plays an important role in the regulation of intercellular communication in plants.Here we describe a simple and fast in vivo staining procedure for the imaging and quantification of callose at PD. We also introduce calloseQuant, a plug-in for semiautomated image analysis and non-biased quantification of callose levels at PD using ImageJ.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

We present a set of extensions to the Free Open Source Software Inkscape that facilitates creation of reproducible figures. Figure panels carry a text description consisting of runnable code that is rendered as figure image panels using widely used image processing, data processing and graphics generation software ImageJ, R, and Processing. A figure saved in the open scalable vector graphics format hence retains important reproducibility information, while its layout and details remain fully customisable.

SummaryThe virome of plants is dominated by RNA viruses1and several of these cause devastating diseases in cultivated plants leading to global crop losses2. To infect plants, RNA viruses engage in complex interactions with compatible plant hosts. In cells at the spreading infection front, RNA viruses replicate their genome through double-stranded RNA (dsRNA) intermediates and interact with cellular transport processes to achieve cell-to-cell movement of replicated genome copies through cell wall channels called plasmodesmata (PD)3. In order to propagate, viruses also must overcome host defense responses. In addition to triggering the antiviral RNA silencing response, RNA virus infection also elicits pattern-triggered immunity (PTI)4whereby dsRNA, a hallmark of virus replication, acts as an important elicitor5. This innate antiviral immune response is also triggered when dsRNA is applied externally and does not require sequence homology to the virus5. However, the mechanism by which PTI restricts virus infection is not known. Here, we show that dsRNA inhibits the progression of virus infection by triggering callose deposition at plasmodesmata and the inhibition of transport through these cell-to-cell communication channels. The dsRNA-induced signaling pathway leading to callose deposition is independent of ROS production and thus distinguished from pathways triggered by bacterial and fungal elicitors. The dsRNA-induced host response at plasmodesmata is suppressed by theTobacco mosaic virusmovement protein (MP). Thus, the virus uses MP to inhibit innate dsRNA-induced immunity at plasmodesmata, which could be a general strategy of phytoviruses to overcome plant defenses and spread infection.