Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically derived oxidation.

Plants are subjected to fluctuations in light intensity, and this might cause unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). Electrons needed for ROS detoxification are drawn, at least partially, from the cellular glutathione (GSH) pool via the ascorbate-glutathione cycle. Here, we explore the dynamics of the chloroplastic glutathione redox potential (chl-EGSH) using high-temporal-resolution monitoring of Arabidopsis (Arabidopsis thaliana) lines expressing the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) in chloroplasts. This was carried out over several days under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-EGSH oxidation during dark-to-light and light-to-dark transitions were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, suggesting two regulated oxidative states of the chl-EGSH. These patterns were not affected in npq1 plants, which are impaired in non-photochemical quenching. Oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulating the chl-EGSH dynamics. Remarkably, pgr5 plants showed an increase in chl-EGSH oxidation during the nights following light stresses, linking daytime photoinhibition and nighttime GSH metabolism. This work provides a systematic view of the dynamics of the in vivo chloroplastic glutathione redox state during varying light conditions.

Distinctive foliar features and volatile profiles in three Ambrosia species (Asteraceae).

MAIN CONCLUSION: Ambrosia species differ both in their trichome types and in metabolic profiles of leaf volatiles. The current study provides tools for easier taxonomic identification of ragweed species. The genus Ambrosia (Asteraceae) includes some of the most noxious allergenic invasive weeds in the world. Due to high polymorphism in this genus, identification of species is often difficult. This study focuses on microscopic investigation of foliar features and GC-MS identification of the main leaf volatile components of three Ambrosia species currently found in Israel-invasive species Ambrosia confertiflora and A. tenuifolia, and transient A. grayi. A. confertiflora and A. tenuifolia have three trichome types: non-glandular trichomes, capitate glandular trichomes and linear glandular trichomes. Their non-glandular trichomes and capitate trichomes have distinct structures and can serve as taxonomic characters. A. grayi (the least successful invader) has only very dense covering trichomes. All three Ambrosia species have secretory structures in their leaf midrib. A. confertiflora, the most problematic invasive plant in Israel, had a ten times higher volatiles content than the other two species. In A. confertiflora, the most abundant volatiles were chrysanthenone (25.5%), borneol (18%), germacrene D and (E)-caryophyllene (both around 12%). In A. tenuifolia, the most abundant volatiles were ß-myrcene (32.9%), (2E)-hexenal (13%) and 1,8-cineole (11.7%). In A. grayi, the most abundant volatiles were ß-myrcene (17.9%), germacrene D (17.8%) and limonene (14%). The three examined species have distinct trichome types and metabolic profiles. Non-glandular trichomes show structural diversification between species and are a good descriptive character. Considering the anthropocentric significance of this highly problematic genus, the current study provides tools for easier identification of ragweed species.

Sensing stress responses in potato with whole-plant redox imaging.

Environmental stresses are among the major factors that limit crop productivity and plant growth. Various nondestructive approaches for monitoring plant stress states have been developed. However, early sensing of the initial biochemical events during stress responses remains a significant challenge. In this work, we established whole-plant redox imaging using potato (Solanum tuberosum) plants expressing a chloroplast-targeted redox-sensitive green fluorescence protein 2 (roGFP2), which reports the glutathione redox potential (EGSH). Ratiometric imaging analysis demonstrated the probe response to redox perturbations induced by H2O2, DTT, or a GSH biosynthesis inhibitor. We mapped alterations in the chloroplast EGSH under several stress conditions including, high-light (HL), cold, and drought. An extremely high increase in chloroplast EGSH was observed under the combination of HL and low temperatures, conditions that specifically induce PSI photoinhibition. Intriguingly, we noted a higher reduced state in newly developed compared with mature leaves under steady-state and stress conditions, suggesting a graded stress sensitivity as part of the plant strategies for coping with stress. The presented observations suggest that whole-plant redox imaging can serve as a powerful tool for the basic understanding of plant stress responses and applied agricultural research, such as toward improving phenotyping capabilities in breeding programs and early detection of stress responses in the field.

Conserved role of matrix metalloproteases 2 and 9 in promoting the migration of neural crest cells in avian and mammalian embryos.

Neural crest cells (NCCs) are a unique embryonic cell population that initially reside at the dorsal neural tube but later migrate in the embryo and differentiate into multiple types of derivatives. To acquire motility, NCCs undergo epithelial-to-mesenchymal transition and invade the surrounding extracellular matrix (ECM). Matrix metalloproteases (MMPs) are a large family of proteases which regulate migration of various embryonic and adult cells via ECM remodeling. The gelatinase's subgroup of MMPs is the most studied one due to its key role in metastasis. As it is composed of only two proteases, MMP2 and MMP9, it is important to understand whether each is indispensable or redundant in its biological function. Here we explored the role of the gelatinases in executing NCC migration, by determining whether MMP2 and/or MMP9 regulate migration across species in singular, combined, or redundant manners. Chick and mouse embryos were utilized to compare expression and activity of both MMPs using genetic and pharmacological approaches in multiple in vivo and ex vivo assays. Both MMPs were found to be expressed and active in mouse and chick NCCs. Inhibition of each MMP was sufficient to prevent NCC migration in both species. Yet, NCC migration was maintained in MMP2-/- or MMP9-/- mouse mutants due to compensation between the gelatinases, but reciprocal pharmacological inhibition in each mutant prevented NCC migration. This study reveals for the first time that both gelatinases are expressed in avian and mammalian NCCs, and demonstrates their fundamental and conserved role in promoting embryonic cell migration.

Altering Neurospora crassa MOB2A exposes its functions in development and affects its interaction with the NDR kinase COT1.

The Neurospora crassa Mps One Binder (MOB) proteins MOB2A and MOB2B physically interact with the Nuclear Dbf2 Related (NDR) kinase COT1 and have been shown to have overlapping functions in various aspects of asexual development. Here, we identified two N. crassa MOB2A residues, Tyr117 and Tyr119, which are potentially phosphorylated. Using phosphomimetic mob-2a mutants we have been able to establish that apart from their previously described roles, MOB2A/B are involved in additional developmental processes. Enhanced conidial germination, accompanied by conidial agglutination, in the phosphomimetic mutants indicated that MOB2A is a negative regulator of germination. Thick-section imaging of perithecia revealed slow maturation and a lack of asci alignment in the mutant strains demonstrating a role for MOB2A in sexual development. We demonstrate that even though MOB2A and MOB2B have some overlapping functions, MOB2B cannot compensate for the roles MOB2A has in conidiation and germination. Altering Tyr residues 117 and 119 impaired the physical interactions between MOB2A and COT1, most likely contributing to some of the observed effects. As cot-1 and the phosphomimetic mutants share an extragenic suppressor (gul-1), we concluded that at least some of the effects imposed by altering Tyr117 and Tyr119 are mediated by the NDR kinase.

Differential regulation of meristem size, morphology and organization by the ERECTA, CLAVATA and class III HD-ZIP pathways.

The shoot apical meristem (SAM) of angiosperm plants is a small, highly organized structure that gives rise to all above-ground organs. The SAM is divided into three functional domains: the central zone (CZ) at the SAM tip harbors the self-renewing pluripotent stem cells and the organizing center, providing daughter cells that are continuously displaced into the interior rib zone (RZ) or the surrounding peripheral zone (PZ), from which organ primordia are initiated. Despite the constant flow of cells from the CZ into the RZ or PZ, and cell recruitment for primordium formation, a stable balance is maintained between the distinct cell populations in the SAM. Here we combined an in-depth phenotypic analysis with a comparative RNA-Seq approach to characterize meristems from selected combinations of clavata3 (clv3), jabba-1D (jba-1D) and erecta (er) mutants of Arabidopsis thaliana We demonstrate that CLV3 restricts meristem expansion along the apical-basal axis, whereas class III HD-ZIP and ER pathways restrict meristem expansion laterally, but in distinct and possibly perpendicular orientations. Our k-means analysis reveals that clv3, jba-1D/+ and er lead to meristem enlargement by affecting different aspects of meristem function; for example, clv3 displays an increase in the stem cell population, whereas jba-1D/+ er exhibits an increase in mitotic activity and in the meristematic cell population. Our analyses demonstrate that a combined genetic and mRNA-Seq comparative approach provides a precise and sensitive method to identify cell type-specific transcriptomes in a small structure, such as the SAM.

Silver Nanoparticles Complexed with Bovine Submaxillary Mucin Possess Strong Antibacterial Activity and Protect against Seedling Infection.

A simple method for the synthesis of nanoparticles (NPs) of silver (Ag) in a matrix of bovine submaxillary mucin (BSM) was reported previously by some of the authors of this study. Based on mucin characteristics such as long-lasting stability, water solubility, and surfactant and adhesive characteristics, we hypothesized that these compounds, named BSM-Ag NPs, may possess favorable properties as potent antimicrobial agents. The goal of this study was to assess whether BSM-Ag NPs possess antibacterial activity, focusing on important plant-pathogenic bacterial strains representing both Gram-negative (Acidovorax and Xanthomonas) and Gram-positive (Clavibacter) genera. Growth inhibition and bactericidal assays, as well as electron microscopic observations, demonstrate that BSM-Ag NPs, at relatively low concentrations of silver, exert strong antimicrobial effects. Moreover, we show that treatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedling transmission of Acidovorax citrulli, one of the most threatening pathogens of cucurbit production worldwide. Overall, our findings demonstrate strong antimicrobial activity of BSM-Ag NPs and their potential application for reducing the spread and establishment of devastating bacterial plant diseases in agriculture.IMPORTANCE Bacterial plant diseases challenge agricultural production, and the means available to manage them are limited. Importantly, many plant-pathogenic bacteria have the ability to colonize seeds, and seed-to-seedling transmission is a critical route by which bacterial plant diseases spread to new regions and countries. The significance of our study resides in the following aspects: (i) the simplicity of the method of BSM-Ag NP synthesis, (ii) the advantageous chemical properties of BSM-Ag NPs, (iii) the strong antibacterial activity of BSM-Ag NPs at relatively low concentrations of silver, and (iv) the fact that, in contrast to most studies on the effects of metal NPs on plant pathogens, the proof of concept for the novel compound is supported by in planta assays. Application of this technology is not limited to agriculture; BSM-Ag NPs potentially could be exploited as a potent antimicrobial agent in a wide range of industrial areas, including medicine, veterinary medicine, cosmetics, textiles, and household products.

Stomatal cell wall composition: distinctive structural patterns associated with different phylogenetic groups.

Background and Aims: Stomatal morphology and function have remained largely conserved throughout ∼400 million years of plant evolution. However, plant cell wall composition has evolved and changed. Here stomatal cell wall composition was investigated in different vascular plant groups in attempt to understand their possible effect on stomatal function. Methods: A renewed look at stomatal cell walls was attempted utilizing digitalized polar microscopy, confocal microscopy, histology and a numerical finite-elements simulation. The six species of vascular plants chosen for this study cover a broad structural, ecophysiological and evolutionary spectrum: ferns ( Asplenium nidus and Platycerium bifurcatum ) and angiosperms ( Arabidopsis thaliana and Commelina erecta ) with kidney-shaped stomata, and grasses (angiosperms, family Poaceae) with dumbbell-shaped stomata ( Sorghum bicolor and Triticum aestivum ). Key Results: Three distinct patterns of cellulose crystallinity in stomatal cell walls were observed: Type I (kidney-shaped stomata, ferns), Type II (kidney-shaped stomata, angiosperms) and Type III (dumbbell-shaped stomata, grasses). The different stomatal cell wall attributes investigated (cellulose crystallinity, pectins, lignin, phenolics) exhibited taxon-specific patterns, with reciprocal substitution of structural elements in the end-walls of kidney-shaped stomata. According to a numerical bio-mechanical model, the end walls of kidney-shaped stomata develop the highest stresses during opening. Conclusions: The data presented demonstrate for the first time the existence of distinct spatial patterns of varying cellulose crystallinity in guard cell walls. It is also highly intriguing that in angiosperms crystalline cellulose appears to have replaced lignin that occurs in the stomatal end-walls of ferns serving a similar wall strengthening function. Such taxon-specific spatial patterns of cell wall components could imply different biomechanical functions, which in turn could be a consequence of differences in environmental selection along the course of plant evolution.

Three-dimensional correlative microscopy of the Drosophila female reproductive tract reveals modes of communication in seminal receptacle sperm storage.

In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues.

Terpenes and cannabidiol against human corona and influenza viruses-Anti-inflammatory and antiviral in vitro evaluation.

The activity of the terpenes and Cannabidiol (CBD) against human coronavirus (HCoV) strain OC43 and influenza A (H1N1) was evaluated in human lung fibroblasts (MRC-5 cells). Also, we examined whether these ingredients inhibit pro-inflammatory cytokines in peripheral blood mononuclear cells (PBMC). The tested preparations exhibited both anti-inflammatory and antiviral effects. The combination of terpenes was effective against both HCoV-OC43 and influenza A (H1N1) virus. The addition of CBD improved the antiviral activity in some, but not all cases. This variation in activity may suggest an antiviral mechanism. In addition, there was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect after 72 h, as observed under a microscope. The anti-inflammatory properties of terpenes were demonstrated using a pro-inflammatory cytokine-inhibition assay, which revealed significant cytokine inhibition and enhanced by the addition of CBD.

Macromolecular sheets direct the morphology and orientation of plate-like biogenic guanine crystals.

Animals precisely control the morphology and assembly of guanine crystals to produce diverse optical phenomena in coloration and vision. However, little is known about how organisms regulate crystallization to produce optically useful morphologies which express highly reflective crystal faces. Guanine crystals form inside iridosome vesicles within chromatophore cells called iridophores. By following iridosome formation in developing scallop eyes, we show that pre-assembled, fibrillar sheets provide an interface for nucleation and direct the orientation of the guanine crystals. The macromolecular sheets cap the (100) faces of immature guanine crystals, inhibiting growth along the π-stacking growth direction. Crystal growth then occurs preferentially along the sheets to generate highly reflective plates. Despite their different physical properties, the morphogenesis of iridosomes bears a striking resemblance to melanosome morphogenesis in vertebrates, where amyloid sheets template melanin deposition. The common control mechanisms for melanin and guanine formation inspire new approaches for manipulating the morphologies and properties of molecular materials.

Zinc chloride is effective as an antibiotic in biofilm prevention following septoplasty.

Biofilm-state bacterial infections associated with inserted medical devices constitute a massive health and financial problem worldwide. Although bacteria exhibit significantly lower susceptibility to antibiotics in the biofilm state, the most common treatment approach still relies on antibiotics, exacerbating the phenomenon of antibiotic-resistant bacteria. In this study, we aimed to assess whether ZnCl2 coating of intranasal silicone splints (ISSs) can reduce the biofilm infections associated with the insertion of these devices and prevent the overuse of antibiotics while minimizing waste, pollution and costs. We tested the ability of ZnCl2 to prevent biofilm formation on ISS both in vitro and in vivo by using the microtiter dish biofilm formation assay, crystal violet staining, and electron and confocal microscopy. We found a significant decrease in biofilm formation between the treatment group and the growth control when ZnCl2-coated splints were placed in patients' nasal flora. According to these results, infections associated with ISS insertion may be prevented by using ZnCl2 coating, thereby obviating the overuse and abuse of antibiotics.

Microscopic and metabolic investigations disclose the factors that lead to skin cracking in chili-type pepper fruit varieties.

The hydrophobic cuticle encasing the fruit skin surface plays critical roles during fruit development and post-harvest. Skin failure often results in the fruit surface cracking and forming a wound-periderm tissue made of suberin and lignin. The factors that make the fruit skin susceptible to cracking have yet to be fully understood. Herein, we investigated two varieties of chili peppers (Capsicum annuum L.), Numex Garnet, whose fruit has intact skin, and Vezena Slatka, whose fruit has cracked skin. Microscopical observations, gas chromatography-mass spectrometry, biochemical and gene expression assays revealed that Vezena Slatka fruit form a thicker cuticle with greater levels of cutin monomers and hydroxycinnamic acids, and highly express key cutin-related genes. The skin of these fruit also had a lower epidermal cell density due to cells with very large perimeters, and highly express genes involved in epidermal cell differentiation. We demonstrate that skin cracking in the Vezena Slatka fruit is accompanied by a spatial accumulation of lignin-like polyphenolic compounds, without the formation of a typical wound-periderm tissues made of suberized cells. Lastly, we establish that skin cracking in chili-type pepper significantly affects fruit quality during post-harvest storage in a temperature-dependent manner. In conclusion, our data highlight cuticle thickness and epidermal cell density as two critical factors determining fruit skin susceptibility to cracking in chili-type pepper fruit.

A green formulation for superhydrophobic coatings based on Pickering emulsion templating for anti-biofilm applications.

This study reports significant steps toward developing anti-biofilm surfaces based on superhydrophobic properties that meet the complex demands of today's food and medical regulations. It presents inverse Pickering emulsions of water in dimethyl carbonate (DMC) stabilized by hydrophobic silica (R202) as a possible food-grade coating formulation and describes its significant passive anti-biofilm properties. The final coatings are formed by applying the emulsions on the target surface, followed by evaporation to form a rough layer. Analysis shows that the final coatings exhibited a Contact Angle (CA) of up to 155° and a Roll-off Angle (RA) lower than 1° on the polypropylene (PP) surface, along with a relatively high light transition. Dissolving polycaprolactone (PCL) into the continuous phase enhanced the average CA and coating uniformity but hindered the anti-biofilm activity and light transmission. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed a uniform coating by a "Swiss-cheese" like structure with high nanoscale and microscale roughness. Biofilm experiments confirm the coating's anti-biofilm abilities that led to the reduction in survival rates of S.aureus and E.coli, by 90-95% respectively, compared to uncoated PP surfaces.

Encapsulation of Bacillus thuringiensis in an inverse Pickering emulsion for pest control applications.

Here, we present an inverse Pickering emulsion-based formulation for Bacillus thuringiensis serovar aizawai (BtA) encapsulations utilized towards pest control applications. The emulsification was carried out by high shear homogenization process via ULTRA-TURRAX®. The water-in-mineral oil emulsions were stabilized by commercial hydrophobic silica. Different silica contents and water/oil ratios were studied. Stable emulsions were obtained at 2 and 3 wt% silica at 30% and 20% water volumes, respectively. The structure of the Pickering emulsions were characterized by laser scanning confocal microscopy and cryogenic scanning electron microscopy. The BtA cells, spores and crystals were encapsulated in the water droplets of the inverse Pickering emulsions. An emulsion composed of 3 wt% silica and 30% water was found to be the most suitable for encapsulation. The pest control efficiency of the encapsulated BtA against Spodoptera littoralis first instar larvae was tested. The studied BtA/emulsion system exhibited a mortality rate of 92%. However, the non-formulated BtA has shown 71% mortality, and the emulsion alone resulted in only 9% mortality. These findings confirm that an emulsion with encapsulated BtA can function as an efficient formulation for biopesticides.

Individual Coating of Entomopathogenic Nematodes with Titania (TiO2) Nanoparticles Based on Oil-in-Water Pickering Emulsion: A New Formulation for Biopesticides.

This study presents a new eco-friendly formulation of entomopathogenic nematodes (EPNs) based on individual coating of EPNs with titanium dioxide (TiO2) nanoparticles (NPs) and mineral oil via oil-in-water Pickering emulsions. Mineral oil-in-water emulsions stabilized by amine-functionalized titanium dioxide (TiO2-NH2) particles were prepared. 40:60 and 50:50 oil-water volume ratios using 2 wt % TiO2-NH2 particles were found to be the most stable emulsions with a droplet size suitable for the formulation and were further studied for their toxicity against the incorporated EPNs. Carboxyfluorescein was covalently bonded to TiO2-NH2 NPs, and the resulting composite was observed via fluorescence confocal microscopy. The dry coating was evaluated using SEM and confocal microscopy, which showed significant nematode coverage by the particles and oil. The final formulation was biocompatible with the studied EPNs, where the viability of the EPNs in the formulation was equivalent to control aqueous suspension after 120 days. Finally, yields of nematodes from infected Galleria mellonella cadavers collected for 150 days showed no significant differences (P > 0.05) using the tested emulsions compared to the control containing nematodes in water.

Platform for Active Vaccine Formulation Using a Two-Mode Enhancement Mechanism of Epitope Presentation by Pickering Emulsion.

The efficiency of epitope-based vaccination (subunit vaccines) is tightly correlated with heterogeneity and the high density of epitope presentation, which maximizes the potential antigenic determinants. Here, we developed a two-mode platform for intensifying the epitope presentation of subunit vaccines. The two-mode epitope presentation enhancement includes a covalent attachment of high concentrations of SARS-CoV-2-S1 peptide epitope to the surface of virus-like-particles (VLPs) and the subsequent assembly of VLP/epitope conjugates on the oil droplet surface at an oil/water interface of an emulsion as Pickering stabilizers. The resultant emulsions were stable for weeks in ambient conditions, and our platform was challenged using the epitope of the SARS-CoV-2-S1 peptide that served as a model epitope in this study. In vivo assays showed that the αSARS-CoV-2-S1 immunoglobulin G (IgG) titers of the studied mouse antisera, developed against the SARS-CoV-2-S1 peptide under different epitope preparation conditions, showed an order of magnitude higher IgG titers in the studied VLP-based emulsions than epitopes dissolved in water and epitopes administered with an adjuvant, thereby confirming the efficacy of the formulation. This VLP-based Pickering emulsion platform is a fully synthetic approach that can be readily applied for vaccine development to a wide range of pathogens.

A standardized nomenclature and atlas of the female terminalia of Drosophila melanogaster.

The model organism Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.