Viable protoplast isolation, organelle visualization and transformation of the globally distributed plant pathogen Phytophthora cinnamomi.

Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics technology has provided significant progress in our understanding of oomycete biology, however, transformation studies of Phytophthora for gene functionalisation are still in their infancy. Only a limited number of Phytophthora species have been successfully transformed and gene edited to elucidate the role of particular genes. There is a need to escalate our efforts to understand molecular processes, gene regulation and infection mechanisms of the pathogen to enable us to develop new disease management strategies. The primary obstacle hindering the advancement of transformation studies in Phytophthora is their challenging and unique nature, coupled with our limited comprehension of why they remain such an intractable system to work with. In this study, we have identified some of the key factors associated with the recalcitrant nature of P. cinnamomi. We have incorporated fluorescence microscopy and flow cytometry along with the organelle-specific dyes, fluorescein diacetate, Hoechst 33342 and MitoTracker™ Red CMXRos, to assess P. cinnamomi-derived protoplast populations. This approach has also provided valuable insights into the broader cell biology of Phytophthora. Furthermore, we have optimized the crucial steps that allow transformation of P. cinnamomi and have generated transformed isolates that express a cyan fluorescent protein, with a transformation efficiency of 19.5%. We therefore provide a platform for these methodologies to be applied for the transformation of other Phytophthora species and pave the way for future gene functionalisation studies.

Chloroplast transformation in new cultivars of tomato through particle bombardment.

A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum L.) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03954-3.

Analysis of heat flow in modified transient plane source (MTPS) measurements of the thermal effusivity and thermal conductivity of materials.

An iterative algorithm for the diffusion of heat in layered structures is solved in cylindrical coordinates for the geometry used in measurements of thermophysical properties of materials by the modified transient plane source (MTPS) method. This solution for the frequency-domain temperature response is then used to model the transient temperature excursion and evaluate the accuracy of the measurements. We evaluate when the MTPS method is capable of separately determining the thermal conductivity and heat capacity per unit volume of a material. For a typical sensor design, data acquisition, and data analysis, the MTPS measurement has a small sensitivity to the thermal diffusivity of the sample when the thermal diffusivity is <5 mm2 s-1. We analyze the propagation of errors from uncertainties in the thermal contact between the sensor and the sample and evaluate the limitations of the MTPS method in accurately measuring samples with extremely low thermal effusivity, e.g., low density foam insulation. We find that uncertainties in the thickness of the contact region limit the accuracy of MTPS measurements when the data are analyzed in a conventional manner based on a single parameter, m-1, the inverse of the slope of the temperature excursion as a function of the square root of time.

Ionic Peltier effect in Li-ion electrolytes.

The coupled transport of charge and heat provide fundamental insights into the microscopic thermodynamics and kinetics of materials. We describe a sensitive ac differential resistance bridge that enables measurements of the temperature difference on two sides of a coin cell with a resolution of better than 10 µK. We use this temperature difference metrology to determine the ionic Peltier coefficients of symmetric Li-ion electrochemical cells as a function of Li salt concentration, solvent composition, electrode material, and temperature. The Peltier coefficients Π are negative, i.e., heat flows in the direction opposite to the drift of Li ions in the applied electric field, large, -Π > 30 kJ mol-1, and increase with increasing temperature at T > 300 K. The Peltier coefficient is approximately constant on time scales that span the characteristic time for mass diffusion across the thickness of the electrolyte, suggesting that heat of transport plays a minor role in comparison to the changes in partial molar entropy of Li at the interface between the electrode and electrolyte. Our work demonstrates a new platform for studying the non-equilibrium thermodynamics of electrochemical cells and provides a window into the transport properties of electrochemical materials through measurements of temperature differences and heat currents that complement traditional measurements of voltages and charge currents.

Boosting Plant Photosynthesis with Carbon Dots: A Critical Review of Performance and Prospects.

Artificially augmented photosynthesis in nano-bionic plants requires tunable nano-antenna structures with physiochemical and optoelectronic properties, as well as unique light conversion capabilities. The use of nanomaterials to promote light capture across photosystems, primarily by carbon dots, has shown promising results in enhancing photosynthesis through tunable uptake, translocation, and biocompatibility. Carbon dots possess the ability to perform both down and up-light conversions, making them effective light promoters for harnessing solar energy beyond visible light wavelengths.This review presents and discusses the recent progress in fabrication, chemistry, and morphology, as well as other properties such as photoluminescence and energy conversion efficiency of nano-antennas based on carbon dots. The performance of artificially boosted photosynthesis is discussed and then correlated with the conversion properties of carbon dots and how they are applied to plant models. The challenges related to the nanomaterial delivery and the performance evaluation practices in modified photosystems, consideration of the reliability of this approach, and the potential avenues for performance improvements through other types of nano-antennas based on alternative nanomaterials are also critically evaluated. It is anticipated that this review will stimulate more high-quality research in plant nano-bionics and provide avenues to enhance photosynthesis for future agricultural applications.

Hybrid achromatic microlenses with high numerical apertures and focusing efficiencies across the visible.

Compact visible wavelength achromats are essential for miniaturized and lightweight optics. However, fabrication of such achromats has proved to be exceptionally challenging. Here, using subsurface 3D printing inside mesoporous hosts we densely integrate aligned refractive and diffractive elements, forming thin high performance hybrid achromatic imaging micro-optics. Focusing efficiencies of 51-70% are achieved for 15µm thick, 90µm diameter, 0.3 numerical aperture microlenses. Chromatic focal length errors of less than 3% allow these microlenses to form high-quality images under broadband illumination (400-700 nm). Numerical apertures upwards of 0.47 are also achieved at the cost of some focusing efficiency, demonstrating the flexibility of this approach. Furthermore, larger area images are reconstructed from an array of hybrid achromatic microlenses, laying the groundwork for achromatic light-field imagers and displays. The presented approach precisely combines optical components within 3D space to achieve thin lens systems with high focusing efficiencies, high numerical apertures, and low chromatic focusing errors, providing a pathway towards achromatic micro-optical systems.

Analysis of plant cuticles and their interactions with agrochemical surfactants using a 3D printed diffusion chamber.

BACKGROUND: Decades of research is available on their effects of single component surfactant on active ingredient diffusion across plant cuticular membranes, but ingredient diffusion is rarely analysed in the presence of commercial surfactants. Also, diffusion studies require expensive or specialized apparatus the  fabrication of which often requires skilled labour and specialized facilities. In this research we have addressed both problems where the effects of four commercially available surfactants on a known tracer molecule were investigated using a 3D printed customized diffusion chamber. RESULTS: As a proof-of-concept a customized 3D printed diffusion chamber was devised using two different thermoplastics and was successfully used in a range of diffusion tests . The effect of various solvents and surfactants on S. lycopersicum cuticular membrane indicated an increased rate of flux of tracer molecules across the membranes. This research has validated the application of 3D printing in diffusion sciences and demonstrated the flexibility and potential of this technique. CONCLUSIONS: Using a 3D printed diffusion apparatus, the effect of commercial surfactants on molecular diffusion through isolated plant membranes was studied. Further, we have included  here the steps involved in material selection, design, fabrication, and post processing procedures for successful recreation of the chamber. The customizability and rapid production process of the 3D printing demonstrates the power of additive manufacturing in the design and use of customizable labware.

Mesoporous silica nanoparticle-induced drought tolerance in Arabidopsis thaliana grown under in vitro conditions.

Nanoparticles of varying formats and functionalities have been shown to modify and enhance plant growth and development. Nanoparticles may also be used to improve crop production and performance, particularly under adverse environmental conditions such as drought. Nanoparticles composed of silicon dioxide, especially those that are mesoporous (mesoporous silica nanoparticles; MSNs), have been shown to be taken up by plants; yet their potential to improve tolerance to abiotic stress has not been thoroughly examined. In this study, a range of concentrations of MSNs (0-5000mgL-1 ) were used to determine their effects, in vitro , on Arabidopsis plants grown under polyethylene glycol (PEG)-simulated drought conditions. Treatment of seeds with MSNs during PEG-simulated drought resulted in higher seed germination and then greater primary root length. However, at the highest tested concentration of 5000mgL-1 , reduced germination was found when seeds were subjected to drought stress. At the optimal concentration of 1500mgL-1 , plants treated with MSNs under non-stressed conditions showed significant increases in root length, number of lateral roots, leaf area and shoot biomass. These findings suggest that MSNs can be used to stimulate plant growth and drought stress tolerance.

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon.

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites. This figure was drawn using BioRender ( https://www.biorender.com ).

Comparative metabolomic profiling of Arabidopsis thaliana roots and leaves reveals complex response mechanisms induced by a seaweed extract.

Seaweed extracts are a prominent class of biostimulants that enhance plant health and tolerance to biotic and abiotic stresses due to their unique bioactive components. However, the mechanisms of action of biostimulants are still unknown. Here, we have used a metabolomic approach, a UHPLC-MS method, to uncover the mechanisms induced following application to Arabidopsis thaliana of a seaweed extract derived from Durvillaea potatorum and Ascophyllum nodosum. We have identified, following the application of the extract, key metabolites and systemic responses in roots and leaves across 3 timepoints (0, 3, 5 days). Significant alterations in metabolite accumulation or reduction were found for those belonging to broad groups of compounds such as lipids, amino acids, and phytohormones; and secondary metabolites such as phenylpropanoids, glucosinolates, and organic acids. Strong accumulations of TCA cycle and N-containing and defensive metabolites such as glucosinolates were also found revealing the enhancement of carbon and nitrogen metabolism and defence systems. Our study has demonstrated that application of seaweed extract dramatically altered the metabolomic profiles of Arabidopsis and revealed differences in roots and leaves that varied across the timepoints tested. We also show clear evidence of systemic responses that were initiated in the roots and resulted in metabolic alterations in the leaves. Collectively, our results suggest that this seaweed extract promotes plant growth and activates defence systems by altering various physiological processes at the individual metabolite level.

Frequency-domain probe beam deflection method for measurement of thermal conductivity of materials on micron length scale.

Time-domain thermoreflectance and frequency-domain thermoreflectance (FDTR) have been widely used for non-contact measurement of anisotropic thermal conductivity of materials with high spatial resolution. However, the requirement of a high thermoreflectance coefficient restricts the choice of metal coating and laser wavelength. The accuracy of the measurement is often limited by the high sensitivity to the radii of the laser beams. We describe an alternative frequency-domain pump-probe technique based on probe beam deflection. The beam deflection is primarily caused by thermoelastic deformation of the sample surface, with a magnitude determined by the thermal expansion coefficient of the bulk material to measure. We derive an analytical solution to the coupled elasticity and heat diffusion equations for periodic heating of a multilayer sample with anisotropic elastic constants, thermal conductivity, and thermal expansion coefficients. In most cases, a simplified model can reliably describe the frequency dependence of the beam deflection signal without knowledge of the elastic constants and thermal expansion coefficients of the material. The magnitude of the probe beam deflection signal is larger than the maximum magnitude achievable by thermoreflectance detection of surface temperatures if the thermal expansion coefficient is greater than 5 × 10-6 K-1. The uncertainty propagated from laser beam radii is smaller than that in FDTR when using a large beam offset. We find a nearly perfect matching of the measured signal and model prediction, and measure thermal conductivities within 6% of accepted values for materials spanning the range of polymers to gold, 0.1-300 W/(m K).

Topological Metal MoP Nanowire for Interconnect.

The increasing resistance of copper (Cu) interconnects for decreasing dimensions is a major challenge in continued downscaling of integrated circuits beyond the 7 nm technology node as it leads to unacceptable signal delays and power consumption in computing. The resistivity of Cu increases due to electron scattering at surfaces and grain boundaries at the nanoscale. Topological semimetals, owing to their topologically protected surface states and suppressed electron backscattering, are promising candidates to potentially replace current Cu interconnects. Here, we report the unprecedented resistivity scaling of topological metal molybdenum phosphide (MoP) nanowires, and it is shown that the resistivity values are superior to those of nanoscale Cu interconnects <500 nm2 cross-section areas. The cohesive energy of MoP suggests better stability against electromigration, enabling a barrier-free design . MoP nanowires are more resistant to surface oxidation than the 20 nm thick Cu. The thermal conductivity of MoP is comparable to those of Ru and Co. Most importantly, it is demonstrated that the dimensional scaling of MoP, in terms of line resistance versus total cross-sectional area, is competitive to those of effective Cu with barrier/liner and barrier-less Ru, suggesting MoP is an attractive alternative for the scaling challenge of Cu interconnects.

Angstrom-scale imaging of magnetization in antiferromagnetic Fe2As via 4D-STEM.

We demonstrate a combination of computational tools and experimental 4D-STEM methods to image the local magnetic moment in antiferromagnetic Fe2As with 6 angstrom spatial resolution. Our techniques utilize magnetic diffraction peaks, common in antiferromagnetic materials, to create imaging modes that directly visualize the magnetic lattice. Using this approach, we show that center-of-mass analysis can determine the local magnetization component in the plane perpendicular to the path of the electron beam. Moreover, we develop Magnstem, a quantum mechanical electron scattering simulation code, to model electron scattering of an angstrom-scale probe from magnetic materials. Using these tools, we identify optimal experimental conditions for separating weak magnetic signals from the much stronger interactions of an angstrom-scale probe with electrostatic potentials. Our techniques should be useful for characterizing the local magnetic order in systems such in thin films, interfaces, and domain boundaries of antiferromagnetic materials, which are difficult to probe with existing methods.

Graphene as a nano-delivery vehicle in agriculture - current knowledge and future prospects.

Graphene has triggered enormous interest in, and exploration of, its applications in diverse areas of science and technology due to its unique properties. While graphene has displayed great potential as a nano-delivery system for drugs and biomolecules in biomedicine, its application as a nanocarrier in agriculture has only begun to be explored. Conventional fertilizers and agricultural delivery systems have a number of disadvantages, such as: fast release of the active ingredient, low delivery efficiency, rapid degradation and low stability that often leads to their over-application and consequent environmental problems. Advanced nano fertilizers with high carrier efficiency and slow and controlled release are now considered the gold standard for promoting agricultural sustainability while protecting the environment. Graphene's attractive properties include large surface area, chemical stability, mechanical stability, tunable surface chemistry and low toxicity making it a promising material on which to base agricultural delivery systems. Recent research has demonstrated considerable success in the use of graphene for agricultural applications, including its utilization as a delivery vehicle for plant nutrients and crop protection agents, as well as in post-harvest management of crops. This review, therefore, presents a comprehensive overview of the current status of graphene-based nanocarriers in agriculture. Additionally, the review outlines the surface functionalization methods used for effective molecular delivery, various strategies for nano-vehicle design and the underlying features necessary for a graphene-based agro-delivery system. Finally, the review discusses directions for further research in optimization of graphene-based nanocarriers.

Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

Nanobionics-Driven Synthesis of Molybdenum Oxide Nanosheets with Tunable Plasmonic Resonances in Visible Light Regions.

Nanobionics-driven synthesis offers a process of designing and synthesizing functional materials on a nanoscale based on the structures and functions of biological systems. An approach such as this is environmentally friendly and sustainable, providing a viable option for synthesizing functional nanomaterials for catalysis and nanoelectronic components. In this work, we present a facile and green nanobionics approach to synthesize plasmonic HxMoO3 by interacting chloroplasts extracted from spinach with two-dimensional (2D) MoO3 nanoflakes. The generated plasmon resonances can be modulated in the visible wavelength ranges, and the efficiency to form the plasmonic materials is enhanced by 90% within 45 min of light excitation compared to reactions without chloroplast involvement. Such a characteristic is ascribed to the interfacial carrier dynamics between the two entities in the reactions, in which highly doped metal oxides with quasi-metallic properties can be formed to generate optical absorptions in the visible light region. The green synthesized plasmonic materials show high photocatalytic activities without the coupling of semiconductors, providing a promising nanoelectronics unit, based on the nanobionics-driven synthesized plasmonic materials.

Odd-even effect on the thermal conductivity of liquid crystalline epoxy resins.

Rapid developments in high-performance computing and high-power electronics are driving needs for highly thermal conductive polymers and their composites for encapsulants and interface materials. However, polymers typically have low thermal conductivities of ∼0.2 W/(m K). We studied the thermal conductivity of a series of epoxy resins cured by one diamine hardener and seven diepoxide monomers with different precise ethylene linker lengths (x = 2-8). We found pronounced odd-even effects of the ethylene linker length on the liquid crystalline order, mass density, and thermal conductivity. Epoxy resins with even x have liquid crystalline structure with the highest density of 1.44 g/cm3 and highest thermal conductivity of 1.0 W/(m K). Epoxy resins with odd x are amorphous with the lowest density of 1.10 g/cm3 and lowest thermal conductivity of 0.17 W/(m K). These findings indicate that controlling precise linker length in dense networks is a powerful route to molecular design of thermally conductive polymers.

Zinc- and magnesium-doped hydroxyapatite-urea nanohybrids enhance wheat growth and nitrogen uptake.

The ongoing and unrestrained application of nitrogen fertilizer to agricultural lands has been directly linked to climate change and reductions in biodiversity. The agricultural sector needs a technological upgrade to adopt sustainable methods for maintaining high yield. We report synthesis of zinc and magnesium doped and undoped hydroxyapatite nanoparticles, and their urea nanohybrids, to sustainably deliver nitrogen to wheat. The urea nanohybrids loaded with up to 42% nitrogen were used as a new source of nitrogen and compared with a conventional urea-based fertilizer for efficient and sufficient nitrogen delivery to pot-grown wheat. Doping with zinc and magnesium manipulated the hydroxyapatite crystallinity for smaller size and higher nitrogen loading capacity. Interestingly, 50% and 25% doses of urea nanohybrids significantly boosted the wheat growth and yield compared with 100% doses of urea fertilizer. In addition, the nutritional elements uptake and grain protein and phospholipid levels were significantly enhanced in wheat treated with nanohybrids. These results demonstrate the potential of the multi-nutrient complexes, the zinc and magnesium doped and undoped hydroxyapatite-urea nanoparticles, as nitrogen delivery agents that reduce nitrogen inputs by at least 50% while maintaining wheat plant growth and nitrogen uptake to the same level as full-dose urea treatments.

High thermal conductivity in wafer-scale cubic silicon carbide crystals.

High thermal conductivity electronic materials are critical components for high-performance electronic and photonic devices as both active functional materials and thermal management materials. We report an isotropic high thermal conductivity exceeding 500 W m-1K-1 at room temperature in high-quality wafer-scale cubic silicon carbide (3C-SiC) crystals, which is the second highest among large crystals (only surpassed by diamond). Furthermore, the corresponding 3C-SiC thin films are found to have record-high in-plane and cross-plane thermal conductivity, even higher than diamond thin films with equivalent thicknesses. Our results resolve a long-standing puzzle that the literature values of thermal conductivity for 3C-SiC are lower than the structurally more complex 6H-SiC. We show that the observed high thermal conductivity in this work arises from the high purity and high crystal quality of 3C-SiC crystals which avoids the exceptionally strong defect-phonon scatterings. Moreover, 3C-SiC is a SiC polytype which can be epitaxially grown on Si. We show that the measured 3C-SiC-Si thermal boundary conductance is among the highest for semiconductor interfaces. These findings provide insights for fundamental phonon transport mechanisms, and suggest that 3C-SiC is an excellent wide-bandgap semiconductor for applications of next-generation power electronics as both active components and substrates.

Metabolites derived from fungi and bacteria suppress in vitro growth of Gnomoniopsis smithogilvyi, a major threat to the global chestnut industry.

INTRODUCTION: Chestnut rot caused by the fungus Gnomoniopsis smithogilvyi is a disease present in the world's major chestnut growing regions. The disease is considered a significant threat to the global production of nuts from the sweet chestnut (Castanea sativa). Conventional fungicides provide some control, but little is known about the potential of biological control agents (BCAs) as alternatives to manage the disease. OBJECTIVE: Evaluate whether formulated BCAs and their secreted metabolites inhibit the in vitro growth of G. smithogilvyi. METHODS: The antifungal potential of BCAs was assessed against the pathogen through an inverted plate assay for volatile compounds (VOCs), a diffusion assay for non-volatile compounds (nVOCs) and in dual culture. Methanolic extracts of nVOCs from the solid medium were further evaluated for their effect on conidia germination and were screened through an LC-MS-based approach for antifungal metabolites. RESULTS: Isolates of Trichoderma spp., derived from the BCAs, significantly suppressed the pathogen through the production of VOCs and nVOCs. The BCA from which Bacillus subtilis was isolated was more effective in growth inhibition through the production of nVOCs. The LC-MS based metabolomics on the nVOCs derived from the BCAs showed the presence of several antifungal compounds. CONCLUSION: The results show that G. smithogilvyi can be effectively controlled by the BCAs tested and that their use may provide a more ecological alternative for managing chestnut rot. The in vitro analysis should now be expanded to the field to assess the effectiveness of these alternatives for chestnut rot management.