High External Concentrations Research Articles

The metal-binding GTPases CobW2 and CobW3 are at the crossroads of zinc and cobalt homeostasis in Cupriavidus metallidurans.

The metal-resistant beta-proteobacterium Cupriavidus metallidurans is also able to survive conditions of metal starvation. We show that zinc-starved cells can substitute some of the required zinc with cobalt but not with nickel ions. The zinc importer ZupT was necessary for this process but was not essential for either zinc or cobalt import. The cellular cobalt content was also influenced by the two COG0523-family proteins, CobW2 and CobW3. Pulse-chase experiments with radioactive and isotope-enriched zinc demonstrated that both proteins interacted with ZupT to control the cellular flow-equilibrium of zinc, a central process of zinc homeostasis. Moreover, an antagonistic interplay of CobW2 and CobW3 in the presence of added cobalt caused a growth defect in mutant cells devoid of the cobalt efflux system DmeF. Full cobalt resistance also required a synergistic interaction of ZupT and DmeF. Thus, the two transporters along with CobW2 and CobW3 interact to control cobalt homeostasis in a process that depends on zinc availability. Because ZupT, CobW2, and CobW3 also direct zinc homeostasis, this process links the control of cobalt and zinc homeostasis, which subsequently protects C. metallidurans against cadmium stress and general metal starvation.IMPORTANCEIn bacterial cells, zinc ions need to be allocated to zinc-dependent proteins without disturbance of this process by other transition metal cations. Under zinc-starvation conditions, C. metallidurans floods the cell with cobalt ions, which protect the cell against cadmium toxicity, help withstand metal starvation, and provide cobalt to metal-promiscuous paralogs of essential zinc-dependent proteins. The number of cobalt ions needs to be carefully controlled to avoid a toxic cobalt overload. This is accomplished by an interplay of the zinc importer ZupT with the COG0523-family proteins, CobW3, and CobW2. At high external cobalt concentrations, this trio of proteins additionally interacts with the cobalt efflux system, DmeF, so that these four proteins form an inextricable link between zinc and cobalt homeostasis.

Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorateaminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5days' post fertilization after neomycin (20μM) and gentamicin (20mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100μM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300μM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20μM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake ofneomycininto hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production andaminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

Modelling of selective ion partitioning between ion-exchange membranes and highly concentrated multi-ionic brines

In recent years, the growing interest in the use of Ion-Exchange Membranes, in the treatment of highly concentrated multi-ionic brines for the selective recovery of critical elements, has prompted the research of fundamental models capable of predicting the IEMs selectivity towards like-charged species. Prior studies have proposed ion partitioning models limited to single-salt solutions that were validated only up to moderate salt concentrations. In this work, we developed a novel multi-ionic extension of the Manning counter-ion condensation model, aiming to predict the sorption selectivity of like-charged counter-ions. Furthermore, the peNRTL model was coupled with the extended Manning model to broaden its applicability range, encompassing membrane equilibrated with very highly concentrated solutions. Novel experimental ion sorption tests with single-salt and binary solutions including NaCl, KCl, MgCl2, and CaCl2 at high concentrations were performed with the commercial cation-exchange membrane Fumasep FKE-50. To the best of Authors’ knowledge, the proposed model for the first time successfully provided quantitative predictions of multi-ionic ion partitioning for all the systems investigated up to extremely high external salt concentrations. The outcomes of this work suggest a strong influence of the local non-electrostatic interactions on the activity coefficients in the membrane phase at high external concentration and highlight the key role of counter-ions hydration state in the condensation phenomenon.

Tartrolon sensing and detoxification by the Listeria monocytogenes timABR resistance operon.

Listeria monocytogenes is a foodborne bacterium that naturally occurs in the soil. Originating from there, it contaminates crops and infects farm animals and their consumption by humans may lead to listeriosis, a systemic life-threatening infectious disease. The adaptation of L. monocytogenes to such contrastive habitats is reflected by the presence of virulence genes for host infection and other genes for survival under environmental conditions. Among the latter are ABC transporters for excretion of antibiotics produced by environmental competitors; however, most of these transporters have not been characterized. Here, we generated a collection of promoter-lacZ fusions for genes encoding ABC-type drug transporters of L. monocytogenes and screened this reporter strain collection for induction using a library of natural compounds produced by various environmental microorganisms. We found that the timABR locus (lmo1964-lmo1962) was induced by the macrodiolide antibiotic tartrolon B, which is synthesized by the soil myxobacterium Sorangium cellulosum. Tartrolon B resistance of L. monocytogenes was dependent on timAB, encoding the ATPase and the permease component of a novel ABC transporter. Moreover, transplantation of timAB was sufficient to confer tartrolon B resistance to Bacillus subtilis. Expression of the timABR locus was found to be auto-repressed by the TimR repressor, whose repressing activity was lost in the presence of tartrolon B. We also demonstrate that tartrolon sensitivity was suppressed by high external potassium concentrations, suggesting that tartrolon acts as potassium ionophore. Our results help to map the ecological interactions of an important human pathogen with its co-residing species within their joint natural reservoir.

Ca2+-Sensitive Potassium Channels.

The Ca2+ ion is used ubiquitously as an intracellular signaling molecule due to its high external and low internal concentration. Many Ca2+-sensing ion channel proteins have evolved to receive and propagate Ca2+ signals. Among them are the Ca2+-activated potassium channels, a large family of potassium channels activated by rises in cytosolic calcium in response to Ca2+ influx via Ca2+-permeable channels that open during the action potential or Ca2+ release from the endoplasmic reticulum. The Ca2+ sensitivity of these channels allows internal Ca2+ to regulate the electrical activity of the cell membrane. Activating these potassium channels controls many physiological processes, from the firing properties of neurons to the control of transmitter release. This review will discuss what is understood about the Ca2+ sensitivity of the two best-studied groups of Ca2+-sensitive potassium channels: large-conductance Ca2+-activated K+ channels, KCa1.1, and small/intermediate-conductance Ca2+-activated K+ channels, KCa2.x/KCa3.1.

Enhanced Pt dispersion and catalytic properties of NaCl-promoted Pt/MFI zeolite catalysts for propane dehydrogenation

A simple and scalable method is presented for preparing propane dehydrogenation catalysts with superior catalytic properties. Pt distribution is significantly improved when the platinum precursor and a large amount of NaCl are introduced into the MFI zeolite by impregnation, and after calcining and fixing the active components in the insoluble form, the excess of inert salt is washed with water. NaCl promotes Pt dispersion, prevents sintering during calcination , and effectively suppresses zeolite acidity. Washing after the calcination of Pt-containing catalysts further increases the catalyst activity and stability due to the redispersion of water-soluble chlorinated Pt compounds. High external surface acid site concentrations, Na/Pt atomic ratios of 40–70, calcination temperatures of 400–500 °C, washing with water, and the order of component introduction when the excess NaCl and the precursor of Pt are calcined jointly contribute to improved Pt dispersion and catalyst stability. • Impregnation-calcination-washing method of Pt catalysts preparation is proposed. • Calcination of the Pt precursor with an excess of NaCl increases the dispersion of Pt. • NaCl excess contributes to stability of propane dehydrogenation MFI-zeolite catalysts. • Washing after calcination improves the activity and stability of Pt/MFI catalysts.

Abscisic acid influences ammonium transport via regulation of kinase CIPK23 and ammonium transporters.

Ammonium uptake at plant roots is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Phosphorylation by the protein kinase calcineurin B-like protein (CBL)-interacting protein kinase 23 (CIPK23) transiently inactivates ammonium transporters (AMT1s), but the phosphatases activating AMT1s remain unknown. Here, we identified the PP2C phosphatase abscisic acid (ABA) insensitive 1 (ABI1) as an activator of AMT1s in Arabidopsis (Arabidopsis thaliana). We showed that high external ammonium concentrations elevate the level of the stress phytohormone ABA, possibly by de-glycosylation. Active ABA was sensed by ABI1-PYR1-like () complexes followed by the inactivation of ABI1, in turn activating CIPK23. Under favorable growth conditions, ABI1 reduced AMT1;1 and AMT1;2 phosphorylation, both by binding and inactivating CIPK23. ABI1 further directly interacted with AMT1;1 and AMT1;2, which would be a prerequisite for dephosphorylation of the transporter by ABI1. Thus, ABI1 is a positive regulator of ammonium uptake, coupling nutrient acquisition to abiotic stress signaling. Elevated ABA reduces ammonium uptake during stress situations, such as ammonium toxicity, whereas ABI1 reactivates AMT1s under favorable growth conditions.

New insights into the role of chrysanthemum calcineurin B\u2013like interacting protein kinase CmCIPK23 in nitrate signaling in Arabidopsis roots

Nitrate is an important source of nitrogen and also acts as a signaling molecule to trigger numerous physiological, growth, and developmental processes throughout the life of the plant. Many nitrate transporters, transcription factors, and protein kinases participate in the regulation of nitrate signaling. Here, we identified a gene encoding the chrysanthemum calcineurin B-like interacting protein kinase CmCIPK23, which participates in nitrate signaling pathways. In Arabidopsis, overexpression of CmCIPK23 significantly decreased lateral root number and length and primary root length compared to the WT when grown on modified Murashige and Skoog medium with KNO3 as the sole nitrogen source (modified MS). The expression of nitrate-responsive genes differed significantly between CmCIPK23-overexpressing Arabidopsis (CmCIPK23-OE) and the WT after nitrate treatment. Nitrate content was significantly lower in CmCIPK23-OE roots, which may have resulted from reduced nitrate uptake at high external nitrate concentrations (≥ 1 mM). Nitrate reductase activity and the expression of nitrate reductase and glutamine synthase genes were lower in CmCIPK23-OE roots. We also found that CmCIPK23 interacted with the transcription factor CmTGA1, whose Arabidopsis homolog regulates the nitrate response. We inferred that CmCIPK23 overexpression influences root development on modified MS medium, as well as root nitrate uptake and assimilation at high external nitrate supply. These findings offer new perspectives on the mechanisms by which the chrysanthemum CBL interacting protein kinase CmCIPK23 influences nitrate signaling.

Flame retardant and mechanical properties of core‐shell‐like polybutylene terephthalate/intumescent flame retardant through controlling injection time

AbstractThis study prepared layered intumescent flame retardant (IFR)‐graphene oxide (GO) via the esterification of GO and IFR. Then the distribution of IFR‐GO in polybutylene terephthalate (PBT) was regulated via an injection molding process, while the tensile strength and combustion were explored. The results showed that the flame retardant distribution in PBT presented high external concentrations and low internal concentrations when the injection time was reduced. The lowest flame retardant content of 20 wt% was evident at an injection time of 2 s while displaying a high limiting oxygen index of 27.1. Although this resulted in a V‐0 flame‐retardant level, the mechanical properties were not affected. Therefore, the flame retardant was proportionately reduced by a shorter injection time to form a concentration gradient core‐shell‐like macrostructure, significantly increasing the flame‐resistant efficiency while retaining the mechanical properties.

Expression of some salt tolerance genes isolated from Egyptian gray mangrove (Avicennia marina)

Mangroves are well-adapted halophytes that thrive in coastal saline environments. They live under difficult environmental conditions, such as high light intensity and external salt concentrations, as well as low-oxygen environments, such as water-logged muck, that are typically inappropriate for the survival of other plants. Salinity is a major abiotic factor that affects plant growth, productivity, and dispersal in tropical and semitropical intertidal areas. Furthermore, it affects approximately 20% of all cultivable land and 50% of all irrigated land on the planet. Mangroves have developed a sophisticated salt filtration mechanism and a complicated root structure to withstand salty water exposure and tidal movement. The expression patterns of five salt tolerance genes (amFer1, amDhna, amSod1, amCat1, and amUbc2) in the Egyptian gray mangrove (Avicennia marina Forssk.) grown under different environmental conditions in South Sinai protectorates (Nabq, Ras Mohamed, Safaga, and Wadi El-Gemal), Egypt, were investigated in this study. This study aimed to assess and examine the genetic behavior of mangroves in response to salinity by using quantitative real-time PCR. Findings revealed differences in the expression patterns of the investigated genes under various conditions, showing that salinity influences plant genetic response. Ferritin gene expression was high in all locations, indicating that ferritin represents an essential component of the mangrove response mechanisms.

Quantal analysis estimates docking site occupancy determining short-term depression at hippocampal glutamatergic synapses.

Before fusion, synaptic vesicles (SVs) pause at discrete release/docking sites. During repetitive stimulation, the probability of site occupancy changes following SV fusion and replenishment. The occupancy probability is considered to be one of the crucial determinants of synaptic strength, but it is difficult to estimate separately because it usually blends with other synaptic parameters. Thus, the contribution of site occupancy to synaptic function, particularly to synaptic depression, remains elusive. Here, we directly estimated the occupancy probability at the hippocampal mossy fibre-CA3 interneuron synapse showing synaptic depression, using statistics of counts of vesicular events detected by deconvolution. We found that this synapse had a particularly high occupancy (∼0.85) with a high release probability of a docked SV (∼0.8) under 3 mm external calcium conditions. Analyses of quantal amplitudes and SV counts indicated that quantal size reduction decreased the amplitudes of all responses in a train to a similar degree, whereas release/docking site number was unchanged during trains, suggesting that quantal size and release/docking site number had little influence on the extent of synaptic depression. Model simulations revealed that the initial occupancy with high release probability and slow replenishment determined the time course of synaptic depression. Consistently, decreasing external calcium concentration reduced both the occupancy and release probability, and the reductions in turn produced less depression. Based on these results, we suggest that the occupancy probability is a crucial determinant of short-term synaptic depression at glutamatergic synapses in the hippocampus. KEY POINTS: The occupancy probability of a release/docking site by a synaptic vesicle at presynaptic terminals is considered to be one of the crucial determinants of synaptic strength, but it is difficult to estimate separately from other synaptic parameters. Here, we directly estimate the occupancy probability at the hippocampal mossy fibre-interneuron synapse using statistics of vesicular events detected by deconvolution. We show that the synapses have particularly high occupancy (0.85) with high release probability (0.8) under high external calcium concentration ([Ca2+ ]o ) conditions, and that both parameter values change with [Ca2+ ]o , shaping synaptic depression. Analyses of the quantal amplitudes and synaptic vesicle counts suggest that quantal sizes and release/docking site number have little influence on the extent of synaptic depression. The results suggest that the occupancy probability is a crucial determinant of short-term synaptic depression at glutamatergic synapses in the hippocampus.

Novel Sources of Variation in Grain Yield, Components and Mineral Traits Identified in Wheat Amphidiploids Derived from Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve (Poaceae) under Saline Soils in India

Salt-affected soils constrain wheat production globally. A wild wheat species, Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve (Poaceae), and its derivatives are tolerant of high external NaCl concentrations but have not been tested yet in field conditions. The aim of this study was to study the performance of amphidiploids derived from T. bessarabicum for grain yield (GYD), yield components and grain mineral composition traits under normal and saline soil conditions. Field experiments were conducted at Karnal (pH(water) = 7.3) and Hisar (pH(water) = 8.3) sites in 2014–2015 and 2015–2016 in India. Grain samples were analysed using inductively coupled plasma–mass spectrometry (ICP-MS). Yield and yield component traits of amphidiploids were typically greater at Karnal than Hisar. The GYD was greater at Karnal (1.6 t ha−1) than Hisar (1.2 t ha−1) in 2014–2015. However, GYD was greater at Hisar (1.7 t ha−1) than Karnal (1.1 t ha−1) in 2015–2016. Mean grain zinc (Zn) concentration of eight amphidiploids, averaged across sites and years, varied from 36 to 43 mg kg−1. Some amphidiploids derived from T. bessarabicum showed greater GYD and grain Zn concentration under saline soils (Hisar) than normal soils (Karnal). These might be potential new sources for the development of salt-tolerant wheat varieties with increased grain Zn concentration under salt-affected soils.

Mechanisms of detoxification of high copper concentrations by the microalga Chlorella sorokiniana.

Microalgae have evolved mechanisms to respond to changes in copper ion availability, which are very important for normal cellular function, to tolerate metal pollution of aquatic ecosystems, and for modulation of copper bioavailability and toxicity to other organisms. Knowledge and application of these mechanisms will benefit the use of microalgae in wastewater processing and biomass production, and the use of copper compounds in the suppression of harmful algal blooms. Here, using electron microscopy, synchrotron radiation-based Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray absorption fine structure spectroscopy, we show that the microalga Chlorella sorokiniana responds promptly to Cu2+ at high non-toxic concentration, by mucilage release, alterations in the architecture of the outer cell wall layer and lipid structures, and polyphosphate accumulation within mucilage matrix. The main route of copper detoxification is by Cu2+ coordination to polyphosphates in penta-coordinated geometry. The sequestrated Cu2+ was accessible and could be released by extracellular chelating agents. Finally, the reduction in Cu2+ to Cu1+ appears also to take place. These findings reveal the biochemical basis of the capacity of microalgae to adapt to high external copper concentrations and to serve as both, sinks and pools of environmental copper.

The functional characterization of LjNRT2.4 indicates a novel, positive role of nitrate for an efficient nodule N2 -fixation activity.

Atmospheric nitrogen (N2) -fixing nodules are formed on the roots of legume plants as result of the symbiotic interaction with rhizobia. Nodule functioning requires high amounts of carbon and energy, and therefore legumes have developed finely tuned mechanisms to cope with changing external environmental conditions, including nutrient availability and flooding. The investigation of the role of nitrate as regulator of the symbiotic N2 fixation has been limited to the inhibitory effects exerted by high external concentrations on nodule formation, development and functioning. We describe a nitrate-dependent route acting at low external concentrations that become crucial in hydroponic conditions to ensure an efficient nodule functionality. Combined genetic, biochemical and molecular studies are used to unravel the novel function of the LjNRT2.4 gene. Two independent null mutants are affected by the nitrate content of nodules, consistent with LjNRT2.4 temporal and spatial profiles of expression. The reduced nodular nitrate content is associated to a strong reduction of nitrogenase activity and a severe N-starvation phenotype observed under hydroponic conditions. We also report the effects of the mutations on the nodular nitric oxide (NO) production and content. We discuss the involvement of LjNRT2.4 in a nitrate-NO respiratory chain taking place in the N2 -fixing nodules.

A Comprehensive Biophysical Model of Ion and Water Transport in Plant Roots. III. Quantifying the Energy Costs of Ion Transport in Salt-Stressed Roots of Arabidopsis.

Salt stress defense mechanisms in plant roots, such as active Na+ efflux and storage, require energy in the form of ATP. Understanding the energy required for these transport mechanisms is an important step toward achieving an understanding of salt tolerance. However, accurate measurements of the fluxes required to estimate these energy costs are difficult to achieve by experimental means. As a result, the magnitude of the energy costs of ion transport in salt-stressed roots relative to the available energy is unclear, as are the relative contributions of different defense mechanisms to the total cost. We used mathematical modeling to address three key questions about the energy costs of ion transport in salt-stressed Arabidopsis roots: are the energy requirements calculated on the basis of flux data feasible; which transport steps are the main contributors to the total energy costs; and which transport processes could be altered to minimize the total energy costs? Using our biophysical model of ion and water transport we calculated the energy expended in the trans-plasma membrane and trans-tonoplast transport of Na+, K+, Cl−, and H+ in different regions of a salt-stressed model Arabidopsis root. Our calculated energy costs exceeded experimental estimates of the energy supplied by root respiration for high external NaCl concentrations. We found that Na+ exclusion from, and Cl− uptake into, the outer root were the major contributors to the total energy expended. Reducing the leakage of Na+ and the active uptake of Cl− across outer root plasma membranes would lower energy costs while enhancing exclusion of these ions. The high energy cost of ion transport in roots demonstrates that the energetic consequences of altering ion transport processes should be considered when attempting to improve salt tolerance.

Co-ion specific effect on sodium halides sorption and transport in a cross-linked poly(p-styrene sulfonate-co-divinylbenzene) for membrane applications

Sodium halide sorption and transport in a cation exchange membrane based on cross-linked poly(p-styrene sulfonate-co-divinylbenzene) (CR61) were investigated experimentally and theoretically. At fixed co-ion valence, sodium halide partitioning between aqueous electrolyte solutions and ion exchange polymers is only minimally sensitive to co-ion chemical identity. NaCl, NaF and NaI exhibit similar solubility coefficients in CR61, which agrees reasonably well with predictions of the Donnan-Manning model, especially at high external salt concentrations. Ion activity coefficients in the membrane were evaluated and ion sorption thermodynamics were investigated. Ion diffusion coefficients in CR61 were predicted using a recently developed framework based on the Manning diffusion model, which accounts for electrostatic effects on ion diffusion, and the Mackie-Meares model, which accounts for tortuosity/area effects on ion diffusion in swollen polymers. Model predictions agree reasonably well with the experimental data, especially at high external salt concentrations. The observed decrease in salt diffusion coefficients in CR61 relative to values in aqueous solutions is due primarily to tortuosity/area effects. Salt diffusion coefficients correlate with co-ion hydrated radius and membrane water content. Finally, salt permeability coefficients predicted using the solution-diffusion model exhibit good agreement with experimental data, especially at external salt concentration greater than 0.1 mol/L.

Evidence that human and equine erythrocytes could have significant roles in the transport and delivery of amino acids to organs and tissues

Erythrocytes have a well-defined role in the gaseous exchange of oxygen and carbon dioxide in the mammalian body. The erythrocytes can contain more than half of the free amino acids present in whole blood. Based on measures showing that venous erythrocyte levels of amino acids are much less than arterial erythrocyte levels, it has previously been proposed that erythrocytes also play a role in the delivery of amino acids to tissues in the body. This role has been dismissed because it has been assumed that to act as an amino acid transport vehicle, the erythrocytes should release their entire amino acid content in the capillary beds at the target tissues with kinetic studies showing that this would take too long to achieve. This investigation set out to investigate whether the equine erythrocytes could rapidly take up and release smaller packages of amino acids when exposed to high or low external concentrations of amino acids, because it seemed very unlikely that cells would be able to release all of their amino acids without serious impacts on osmotic balance. Freshly prepared erythrocytes were placed in alternating solutions of high and low amino acid concentrations in PBS to assess the capacities of these cells to rapidly take up and release amino acids depending on the nature of the external environment. It was found that amino acids were rapidly taken up and released in small quantities in each cycle representing 15% of their total load in equine erythrocytes and 16% in human erythrocytes. The capacity for rapid uptake/release of amino acids by equine and human erythrocytes provided evidence to support the theory that mammalian erythrocytes have a significant role in transport of amino acids from the liver to tissues, muscles and organs.

Transcriptome analysis of Medicago lupulina seedlings leaves treated by high calcium provides insights into calcium oxalate formation

Calcium oxalate (CaOx) is a common biomineral found in the plant kingdom. Crystals of CaOx occur in different plant tissues, such as leaves and stems. However, little is known about the biosynthesis of CaOx in oxalate-accumulating plants. Moreover, the literature on genes related to CaOx formation under high-calcium environment is scarce. In the present study, the physiological parameters and the transcript profiles of Medicago lupulina leaves treated with 0.1 and 25 mM Ca2+ were analyzed to study the genes involved in the biosynthesis of CaOx. We demonstrated that exposure to high external calcium concentration induced H2O2 production, ascorbic acid degradation, and CaOx accumulation in M. lupulina leaves. Moreover, we identified 1715 differentially expressed genes (DEGs) (1322 up-regulated and 393 down-regulated genes) in leaves treated with 25 mM Ca2+ compared with the leaves treated with 0.1 mM Ca2+. We further demonstrated the involvement of DEGs in oxalic acid production, calcium transport, and calcium buffering. These results revealed that a high calcium promoted oxalic acid biosynthesis by inducing the expression of NADPH oxidase and ascorbate oxidase genes. In addition, several genes encoding cyclic nucleotide-gated channel, Ca2+-ATPase, H+/Ca2+ exchangers, and calcium-binding proteins were found to be differentially expressed and involved in calcium transport and calcium buffering. Our transcriptome analyses provide a comprehensive insight into the biosynthesis of CaOx in oxalate-accumulating plants.

Thermodynamic analysis of the pathway for ethanol production from cellobiose in Clostridium thermocellum

Clostridium thermocellum is a candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts the maximum ethanol titer achieved to date remains below industrially required targets. Several studies have analyzed the impact of increasing ethanol concentration on C. thermocellum’s membrane properties, cofactor pool ratios, and altered enzyme regulation. In this study, we explore the extent to which thermodynamic equilibrium limits maximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014) to identify the range of allowable metabolite concentrations that maintain a negative free energy change for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a time-series metabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamic bottlenecks under high external ethanol concentrations. Thermodynamic analysis was also deployed in a prospective mode to evaluate genetic interventions which can improve pathway thermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) which possess unique genetic variations while ensuring mass and redox balance with ethanol production. MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK) has a higher pathway MDF than the malate shunt alternative due to limiting CO2 concentrations under physiological conditions, and ii) NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) can alleviate thermodynamic bottlenecks at high ethanol concentrations due to cofactor modification and reduction in ATP generation. The combination of ATP linked phosphofructokinase (PFK-ATP) and NADPH linked alcohol dehydrogenase (ADH-NADPH) with NADPH linked aldehyde dehydrogenase (ALDH-NADPH) or ferredoxin: NADP ​+ ​oxidoreductase (NADPH-FNOR) emerges as the best intervention strategy for ethanol production that balances MDF improvements with ATP generation, and appears to functionally reproduce the pathway employed by the ethanologen Thermoanaerobacterium saccharolyticum. Expanding the list of measured intracellular metabolites and improving the quantification accuracy of measurements was found to improve the fidelity of pathway thermodynamics analysis in C. thermocellum. This study demonstrates even before addressing an organism’s enzyme kinetics and allosteric regulations, pathway thermodynamics can flag pathway bottlenecks and identify testable strategies for enhancing pathway thermodynamic feasibility and function.

Cellular distribution of cadmium in two amaranth (Amaranthus mangostanus L.) cultivars differing in cadmium accumulation.

Differences in cellular cadmium (Cd) distribution between Cd-tolerant and Cd-sensitive lines of amaranth (Amaranthus mangostanus L.) may reveal mechanisms involved in Cd tolerance and hyperaccumulation. We compared the cellular distribution and accumulation of Cd in roots, stems, and leaves between a low-Cd accumulating cultivar (Zibeixian, L-Cd) and a high-Cd accumulating cultivar (Tianxingmi, H-Cd) in a hydroponic experimental system. In all treatments, H-Cd grew better than L-Cd and accumulated more Cd. As the Cd concentration increased, the H-Cd plants grew normally and their biomass increased, except in the 60 μM Cd treatment. The biomass of L-Cd decreased with increasing Cd concentrations. The highest Cd concentration in the roots, stems, and leaves of H-Cd was 950 mg/kg, 305 mg/kg, and 205 mg/kg, respectively, compared with 269 mg/kg, 62.9 mg/kg, and 74.8 mg/kg, respectively, in L-Cd. The Cd distribution differed between the two cultivars. Scanning and transmission electron microscopy and energy-dispersive spectrometry analyses showed that Cd was distributed across the entire cross section of H-Cd roots but largely restricted to the epidermal cells and the exodermis of L-Cd roots. The main Cd storage sites were the root apoplast, cell walls, and intercellular spaces in H-Cd and the root epidermal cells and the exodermis in L-Cd. In H-Cd leaves, Cd accumulated mainly in vacuoles of epidermal cells and, at high external Cd concentrations, in the vacuoles of mesophyll cells.