Recent studies have reported conflicting findings on the effect of OA on growth and N2 fixation of Trichodesmium. Here, we quantitatively analyzed experimental data on how Trichodesmium reallocated intracellular iron and energy among key cellular processes in response to OA, and integrated the findings to construct an optimality-based cellular model. The model results indicate that Trichodesmium growth rate decreases under OA primarily due to reduced nitrogenase efficiency. The downregulation of the carbon dioxide (CO2)-concentrating mechanism under OA has little impact on Trichodesmium, and the energy demand of anti-stress responses to OA has a moderate negative effect. We predict that if anthropogenic CO2 emissions continue to rise, OA could reduce global N2 fixation potential of Trichodesmium by 27% in this century, with the largest decrease in iron-limiting regions.The response of the prominent marine dinitrogen  on the growth and N It is thus important to understand how N2 fixation will respond to ocean acidification  caused by the dissolution of anthropogenic CO2 in the ocean2. Most previous studies have shown that the growth and N2 fixation of marine diazotrophs, particularly the prominent genus Trichodesmium, increased with OA3. In contrast, recent studies have reported no significant or even negative effects of OA on diazotrophs7.Marine N2-concentrating mechanisms (CCM) under high CO2 concentration, which seemingly saves energetic resources for other cellular processes including N2 fixation11. Hong et al.7, however, reported that OA inhibited the growth and N2 fixation of Trichodesmium, because the beneficial effect of high CO2 concentration was overwhelmed by the negative effect of low pH. Their study suggested that Trichodesmium needed to invest additional cellular resources and energy to cope with the stress imposed by low pH . Regardless of whether OA effects are positive or negative, these studies all highlighted the importance of energy metabolism in the response of Trichodesmium to OA, even though they lacked a quantitative understanding of the energy budget.The growth enhancement of diazotrophs under OA is often attributed to the downregulation of COTrichodesmium and is often a limiting resource for the diazotroph in a large part of the ocean14. Trichodesmium normally allocates a significant portion of intracellular Fe to nitrogenase, an Fe-rich enzyme that catalyzes N2 fixation, and the remaining Fe is used in photosystems and other cellular processes18. It has been shown that Trichodesmium can reallocate intracellular Fe among different cellular processes in response to Fe limitation. For example, under Fe deficiency Trichodesmium can compromise on N2 fixation to conserve Fe for photosynthesis20. Conversely, Fe can be reallocated from photosystems to nitrogenase in Fe-limited Trichodesmium to compensate for the decreased nitrogenase efficiency under OA7. However, it is not fully understood how the reallocation of Fe and energy are quantitatively linked. There still exists a gap between experimental results and model predictions of N2 fixation in the future acidified ocean.Iron (Fe) plays a vital role in energy metabolism of Trichodesmium in response to OA and examine how they modulate its growth and N2 fixation. The results of these quantitative analyses provide parameterization schemes for an optimality-based Trichodesmium cellular model, in which growth rate is maximized by optimizing allocation of intracellular Fe and energy under varying levels of OA and intracellular Fe. By using the model to study different physiological processes, we find that the reduced nitrogenase efficiency dominates the response of Trichodesmium to OA. Furthermore, we project that N2 fixation potential by Trichodesmium in the global ocean may be reduced by 27% by the end of this century if anthropogenic CO2 emissions continue to rise.In this study, using experimentally-measured intracellular Fe in photosystems and nitrogenase together with other parameters obtained from the literature, we quantitatively analyze intracellular Fe and energy allocations in Trichodesmium cellular model in which N2 fixation is the only source of N for the diazotroph  is allocated to CCM (ECCM), carbon (C) fixation (ECF), N2 fixation (ENF), maintenance (EMT) and anti-stress against OA (EAts) . In this model, the amount of Fe allocated to photosystems determines the energy production rate, and the subsequent allocation of energy determines the C fixation rate. The N2 fixation rate also depends on the energy allocated to this process and the amount of Fe allocated to nitrogenase. Therefore, at a given condition, the model potentially has a solution for optimal allocation of intracellular Fe and energy for maximal growth, under which the ratio of C to N fixation equals the elemental stoichiometry of Trichodesmium cells and no intracellular Fe and energy are wasted.In the model framework Fig. , the totmentclass2pt{minimQFe, To resolve different intracellular Fe pools, mentclass2pt{minimMT, ratio of carbon-based specific growth rate gc to 7; and third, comparing high Fe to low Fe conditions, the expression level of Fe-containing proteins involved in maintenance increased relatively less than the increase in growth rate . As QFe, see Table MT as a constant parameter to calculate We first estimated mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimQFe), which was consistent with our observation that QFe in Trichodesmium grown at even moderate rates was markedly higher than the Fe in nitrogenase and photosystems in those grown at the highest rates  (intermediate Fe limitation) result in lesser increases in growth rate. We interpret these earlier data to suggest that only a small portion of the marginal increase in QFe is used for growth and that perhaps 80% of this marginal increase is used for storage; this interpretation is consistent with our model results. Although such high Trichodesmium given the high Fe storing capacity of Dps23 and ferritin25  (Supplementary Tables With mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimf ST) of excess cellular Fe  than in the high-Fe treatment (24%)  Table  −1  to increase NifH (the nitrogenase reductase of the nitrogenase complex) by 47% at mid-day but by 37% on average on a daily basis4.To match the observed N)−1 Fig. . HoweverTrichodesmium CCM, we considered a simple scheme including that first, a portion (f BC) of total inorganic carbon uptake (Ci) into the cell is from energy-consuming bicarbonate (HCO3−) transport; second, passive CO2 diffusion contributes the rest of Ci; and third, a portion (lk) of Ci leaks out of the cell as CO2  dehydrogenase complex located at the thylakoid membrane28. The mechanistic details of this system have not been determined, but the overall energy cost is thought to be small. NADPH consumption supporting proton removal is likely coupled to ATP generation resulting in a low net energy cost29. We thus neglect this CO2 uptake dependent energy consumption and only consider the energy consumption associated with HCO3− transport. Transport of 1 mol HCO3− costs 1 mol ATP30 (approximately 50 kJ31), and building HCO3− transporters and carboxysomes also requires energy, which, however, is hard to quantify and is assumed to cost additional 20% energy. Altogether the cost is estimated at 60 kJ per mol HCO3− transported. Using f BC = 80% and lk = 50% in Trichodesmium33, 1 mol C fixation requires 2.0 mol Ci, in which 1.6 mol is from HCO3− transport, and accordingly 96 kJ of energy , the CCM energy cost rate would increase from 96 to 160 kJ (mol C)−1, which is still small compared to the total energy cost ems Fig. . This waQFe and seawater pCO2 and pH, Trichodesmium growth and N2 fixation rates in the model are a function of only two parameters, i.e., The parameterization schemes established above were integrated into the cellular model framework Fig. . As show2 fixation rates increase with QFe. Above the Fe threshold (at which the cells start to store Fe) the rate of increase is reduced  and QFe show that OA has a large impact on Trichodesmium when QFe is low −1, OA changes Trichodesmium growth rate by no more than ±20% with a pH change of ±0.2 unit  show that within a QFe range of 20–300 μmol Fe (mol C)−1, CCM downregulation alone increases Trichodesmium growth rate by only ~0.6%, while anti-stress energy consumption and reduced nitrogenase efficiency decrease growth by ~11% and 18–46%, respectively . The simulated optimal reallocations of the intracellular resources demonstrate that when the anti-stress energy consumption is the only OA effect, a relatively small portion of nitrogenase Fe (~15%) is reallocated to the photosystems, consequently increasing photosystem Fe greatly by ~40%  shows a strong linear relationship (R2 = 0.65), with the regression line [Log(QFe) = −0.17 + 0.83Log(Fe′)] close to that obtained from a laboratory experiment4  mmol N (mol C)−1 d−1, with the highest potential in the oceanic regions near the Indonesian archipelago and north of Australia and the lowest potential in the southeastern and northeastern subtropical Pacific  under the Representative Concentration Pathway (RCP) 8.5, a scenario in which anthropogenic greenhouse gas (including CO2) emissions continue to rise throughout the 21st century , which can be determined by optimally allocating intracellular Fe and energy to maximize the growth rate of Trichodesmium. The results demonstrated that Trichodesmium cannot cope with the cumulative stresses imposed by OA and hence may decline in a future, more acidic ocean.In the present study, we systematically and quantitatively evaluated the impact of OA on the prominent marine diazotroph 2 fixation rates, relied mainly on the values of those parameters explicitly or implicitly related to energy consumption, IUE, and the strength of the OA impacts. The major consumption of cellular energy by C and N2 fixation was constrained by free energy changes of those biochemical reactions   in Trichodesmium, even not to mention the energy requirement for coping with the negative impacts of OA. Thus, the positive effect of OA reported previously is likely caused by some of the artificial growth conditions applied that supported overall suboptimal growth rates and potentially altered key physiological processes in Trichodesmium7.The modeled optimal allocations of intracellular Fe and energy, and therefore the modeled maximal growth and Nns Table , which iO2) Fig. , which i+], which however remains to be verified. Nevertheless, as discussed above, the model was not very sensitive to the rate of the anti-stress energy consumption as energy can be generated to meet this need by reallocating a small amount of cellular Fe to the photosystems. This, however, assumed that light was not limiting. Our model experiment showed that when cellular energy production is light-limited (see Methods), the anti-stress energy requirement would further reduce Trichodesmium growth rate at high intracellular Fe , when the Fe supply is high, likely reflect its response to natural fluctuations in Fe availability by storing Fe for later use. On a longer time-scale, it is possible that the diazotroph may evolve to optimally allocate its cellular Fe, investing more in metabolism than in storage to compensate for the reduced nitrogenase efficiency. This phenomenon has already been found in our study where the Fe storage started at a higher threshold of intracellular Fe  under OA  under ambient  and acidified conditions  are obtained from Hong et al.7, in which Trichodesmium were pre-acclimated to experimental conditions for 2–3 months before the rates were measured. Inorganic Fe concentration (Fe′) (pM) was estimated from FeT, pH, light, temperature, and concentration of the chelating agent EDTA in the medium47. The intracellular Fe quota (QFe) [μmol Fe (mol C)−1] was calculated based on a regression between the logarithms of Fe′ and QFe 4, which is observed previously with the same organism cultured under the same experimental conditions as in the present study.Growth and N4, cells from each one of the triplicate cultures grown under different conditions  were collected at midday of the photoperiod for quantifying expression of nitrogenase and photosynthetic Fe-containing proteins including: NifH, PsbA, the D1 protein of photosystem II (PSII), PetC, a key subunit of cytochrome (cyt) b6/f complex, and PsaC, the core subunit of photosystem I (PSI), in unit of μmol per g total cellular protein, by immunoblot analyses. Briefly, steady-stately growing cells were collected by filtration onto 3 µm polycarbonate membrane filters (Millipore), flash frozen in liquid nitrogen, and then stored at −80 °C for later analysis. Proteins were extracted and denatured in an extraction buffer  with heating at 100 °C for 10 min. Insoluble material was pelleted by centrifugation at 12,000 g for 10 min, and total protein in the supernatant was quantified using the bicinchoninic acid (BCA) assay . Equivalent amounts of total protein  was separated on a 12% SDS polyacrylamide gel for 20 min at 80 V followed by 60 min at 120 V in 1 × SDS running buffer, and then transferred onto a PVDF membrane in ice-cold transfer buffer  for 20 min at 300 mA. The membrane was then blocked for 1 h in TBST buffer  containing 5% nonfat milk, followed by 1–2 h incubation with primary antibody  and subsequently three 10-min washes with TBST buffer. The membrane was then probed with alkaline phosphatase-conjugated goat anti-rabbit IgG or anti-chicken IgY for 1 h or 2 h, respectively, and washed three times again. Following three rinses with PhoA buffer , protein bands on the membrane were visualized with NBT/BCIP  and quantified by densitometry.Following Shi et al.−1] was estimated to be 38/4 Fe (NifH)−1 −1], and the sum of Fe in these proteins gave an estimate of Fe in photosystems −1, or 7.2 g protein (mol C)−1, was applied to convert the estimates to Fe quota [μmol Fe (mol C)−1] QFe, pH and pCO2:Model parameterization schemes based on the quantitative analyses were integrated to a model only depending on two unknown parameters Here we briefly describe the model equations. Description and values of all the model parameters will not be included here but are listed in Table QFe for Fe storage is:+ concentration to a baseline condition The threshold of gC) from the energy consumption rate of carbon fixation:gC. The energy consumption for the CCM is:2 fixation:2 fixation rate from energy consumption rate of N2 fixation:gC :2 fixation:2 fixation rate can be estimated by the following two equations accounting for effect of 2 fixation rate is the smaller of NFE and NFFe. The N-based growth rate gN can be calculated by multiplying the N2 fixation rate with a molar C:N of 5.6, an average value obtained from our culture experiments. Then the modeled Trichodesmium growth rate, g, is the smaller of gC and gN.Hence the residual Fe is subsequently allocated to N48 is used to solve Eq. (g.MATLAB optimization function fminsearcholve Eq.  by optimpCO2 levels as the ambient and acidified conditions of the culture experiments. The minimum intracellular Fe level of the simulation is 20 μmol Fe (mol C)−1, as from our culture experiments the diazotroph cannot survive below this level. The model is then simulated for intracellular Fe level of 20–300 μmol (mol C)−1 and environmental pH of 7.75–8.20, with pCO2 determined from varying pH and a constant alkalinity of 2200 μmol kg−1 under salinity of 35 PSU and temperature of 35 °C using the CO2SYS program (http://cdiac.ornl.gov/ftp/co2sys/)49.To compare with the observations, the model first runs at the same pH and QFe range of 20–300 μmol Fe (mol C)−1 at two pH levels of 8.02 and 7.82, with the pCO2 determined in same way as used above. The relative change of the modeled growth rate at the two pH levels was calculated for each experiment. The relative changes of Fe in nitrogenase and photosystems and energy production rate were also calculated for the anti-stress energy consumption only and the reduced nitrogenase efficiency only experiments.In each experiment, the model only enabled one OA effect: the reduced CCM energy consumption, the anti-stress energy consumption under lower pH or the reduced nitrogenase efficiency. Each experiment run over a −1 d−1, a rate that corresponds to the ambient high-Fe treatment of the culture experiment, and the anti-stress energy consumption was enabled as the only OA effect.A light-limiting experiment was set up in which the maximum energy production rate is 560 kJ (mol C)Trichodesmium’s potential to fix N2 (per Trichodesmium C biomass per unit time) solely determined by Fe and pH/pCO2. We first collected in situ Trichodesmium Fe quota (QFe) measurements from literature55, and binned them according to their sampling location (1° × 1°) and time (month)  and Log(Fe′), in which a data source54 was excluded because it used a different method from and obtained much lower QFe than other studies, and therefore may not be directly comparable to other data sources. The obtained regression between Log(QFe) and Log(Fe′) was then used to estimate Trichodesmium QFe from the CESM-BGC surface Fe′ in 1990s between 35°S and 35°N, a probable range that Trichodesmium may exist58.Our study projects pCO2 in 1990s and 2090s  at Supplementary InformationPeer Review File


Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles (selected from publicly available Enamine Ltd. Screening Collection) in thermal shift assay against bovine carbonic anhydrase ( Earlier, we conducted a high-throughput DSF screening of a chemically diverse set of 8000 compounds selected from the Enamine screening collection comprising over 2,000,000 compoundsbCA), a protein most closely resembling isoform II of the human carbonic anhydrase (hCA II). This led to the discovery of three compounds (1–3) that produced noticeable thermal shift of bCA mT (ΔmT) which did not belong to any of then-known classes of carbonic anhydrase inhibitors and which we dubbed as Strecker α-aminonitriles considering they could be obtained from various ketones via the Strecker reaction. Besides the TSA biophysical readout, these compounds were found, by testing in biochemical stopped-flow kinetics CO2 hydration assay, to inhibit hCA II  in the single-digit micromolar range , also termed thermal shift assay (TSA), is an efficient technique for direct determination of a small molecule’s affinity to a protein targetar range 3.1–3 acted as true inhibitors (and sources of adventitious cyanide anion which is known to inhibit hCA), we postulated that α-aminonitriles in general may act as suicide in situ donors of CN- anion  can be expected from any representatives of this chemotype with sufficient complementarity (affinity) to the enzyme’s active site. In order to verify this hypothesis and also to discover more potent inhibitors belonging to this class of compounds as well as to establish structure-activity relationships (SAR), we undertook more focussed screening of 800 publicly available α-aminonitriles selected from the Enamine Ltd. screening collectionhCA II, an established target for glaucoma and diuretic drugsAs it was confirmed that compounds Supplemental Material) for the follow-up DSF screening were selected by substructure search and obtained directly from the Enamine screening collection1H NMR spectroscopy prior to biochemical testing in CA inhibition assay.The 800 α-aminonitriles bCA binder at 10 and 20 µM concentration.Thermal shift assay was carried out using ViiA™ 7 Real-Time PCR System equipped with 384-well block . The TSA procedure was adopted from the literature2PO4/Na2HPO4, pH 7.0, 10 mM NaCl. The total volume of the reaction mixture per well was 10 µL. Carbonic anhydrase (Sigma Aldrich Cat# C3934) in 300 µg/mL concentration was pre-mixed with environment-sensitive SYPRO orange dye  at final concentration in the reaction of 10 ×, with regard to the stock concentration stated by the vendor. The mixtures were pre-incubated for 1 h at 4 °C with 20 µM concentrations of the compounds , placed into MicroAmp® Optical 384-Well Reaction Plate . The reaction mixture was kept at room temperature for 15 min to ensure full protein-compound interactions. The temperature was raised at 1.6 °C/s rate to 40 °C without signal reading. Starting from 40 °C up to 85 °C the heating rate was set to 0.05 °C/s with constant fluorescence reading, using 470/623 nm filter set. The raw data of dye fluorescence intensity change upon melting of the protein were obtained from the instrument ViiA 7 RUO software. Further data processing and visualisation was performed by custom-made Microsoft Excel scripts. The peak of the first derivative for the fluorescence curve was used to define melting temperature mT. mT for DMSO control wells, having only protein, dye and 1% DMSO was used as a oT to determine melting temperature shifts (ΔmT). All measurements were made in quadruplicates.For 800-compound screening, the test reactions were set up in the following buffer: 10 mM NaH2 hydration activity2SO4 (for maintaining constant ionic strength), following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The non-catalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were combined and pre-incubated for 15 min at room temperature prior to running the assay, in order to allow for the formation of the ezyme–inhibitor complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlierhCA II was obtained in-house as reported earlierAn Applied Photophysics stopped-flow instrument was used for assaying the CA catalysed COSupplemental Data) from the Enamine, Ltd. Screening CollectionbCA for the ability to shift the enzyme’s melting temperature (mT) using acetazolamide (AZ)bCA mTConsidering that amino acetonitrile moiety (characteristic of the Strecker-type α-aminonitrile chemotype) was postulated as the pharmacophore for the recently discoveredmT,°C) as well as the quality of the melting curve obtained  were taken into account. With these two criteria, a total of 47 compounds were selected and tested in stopped-flow CO2 hydration assay for their ability to inhibit hCA II. The iK values presented in hCA II.As it follows from the graphical representation of the TSA screening data obtained for the 800 compounds , none ofhCA II inhibition in the same single-digit micromolar iK range as the initial hits (1–3). Reassuringly, however, seven compounds displayed iK values in the submicromolar range, which represented a 10-fold improvement compared to inhibitory potency of 1–3 towards hCA II. Moreover, these compounds, while belonging to the general Strecker α-aminonitrile class, turned out to be structurally distinct from the initial hits 1–3 as they all are based on a new N-(cyanomethyl)piperazine scaffold  bear a hydrophobic aliphatic or aromatic substituent on the same carbon atom as the cyano group, the 4N position of the piperazine ring is substituted with a relatively polar group. Moreover, examination of the SAR information contained in the total TSA screening data , reveals that hydrophobic groups at 4N reduce the compound’s affinity to the target (as indirectly measured by ΔmT). Such an active chemotype topology appear to be in line with the known distinct architecture of the carbonic anhydrase active site where two halves – hydrophobic and hydrophilic are clearly delineatedin situIt is also evident that while all frontrunner compounds (bCA) led not only to further validation of this new class of inhibitors but also to refinement of the active chemotype, which became possible after biochemical testing of 47 selected TSA hits for hCA II inhibition in stopped-flow CO2 hydration assay. The active chemotype can be defined as 1N-(cyanomethyl)piperazine bearing two other substituents (hydrophilic and hydrophobic) on the opposite sides of the piperazine core. Such a dichotomy of the newly identified pharmacophore appears to be in line with the known bipolar character of the enzyme’s active site. Further studies are underway to investigate the validity of this hypothesis.Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles in thermal shift assay against bovine carbonic anhydrase (Click here for additional data file.


It was clarified by this study that limited ductility of hexagonal Mg alloys could be overcome by activating unusual slip systems (<c + a> dislocations) in fully recrystallized UFG microstructures.Recently, we have found that fully recrystallized ultrafine-grained (UFG) microstructures could be realized in a commercial precipitation-hardened Magnesium (Mg) alloy. The UFG specimens exhibited high strength and large ductility under tensile test, but underlying mechanisms for good mechanical properties remained unclear. In this study, we have carried out systematic observations of deformation microstructures for revealing the influence of grain size on the change of dominant deformation modes. We found that plastic deformation of conventionally coarse-grained specimen was predominated by {0001} <11–20> slip and {10–12} <10–11> twinning, and the quick decrease of work-hardening rate was mainly due to the early saturation of deformation twins. For the UFG specimens, {10–12} <10–11> twinning was dramatically suppressed, while non-basal slip systems containing < However, so far, the applications of Mg and Mg alloys are substantially restricted and most of them are used in cast products. One of the reasons to hinder their widespread applications is the low strength, which is much lower than that of other metallic materials widely used. Another critical shortcoming is their poor ductility and formability. Unlike other metallic materials, such as steels and Al alloys having cubic crystal structures, Mg has a hexagonal close-packed (HCP) crystal structure with an axial ratio (c/a) of 1.624. As a result, only {0001} <11–20> basal slip system having <a> Burgers vector is easily activated at room temperature. However, basal slip can realize only 2-dimentional deformation in each grain, which is far less than the 5 independent slip systems required for free deformation in polycrystalline materials in von Mises criterion, resulting in limited cold-forming capability as well as pronounced mechanical anisotropy in Mg alloys8. Deformation twinning is another important deformation mechanism in addition to basal slip, which can accommodate strains along the c-axis during plastic deformation10. However, the deformation twins usually induce the formation of cracks or voids at grain boundaries they impinge, leading to failure11. Therefore, a lot of efforts have been paid to improve the strength and ductility/formability in Mg alloys.As the lightest metal, magnesium (Mg) and its alloys have long been considered as candidate materials to be used in aerospace and automobile industries12. However, high temperature processes cost and may lead to grain coarsening. Furthermore, intermediate annealing is often required during multi-pass high temperature deformation to avoid cracks, which increases the processing cost furthermore.Processing at elevated temperatures is one possible solution for improving the formability of Mg alloys, since multiple slip systems in addition to basal slip can be activated at elevated temperatures because of the temperature dependence of critical resolved shear stress (CRSS) for each slip system15. For instance, Mukai et al.15 found that the grain size of a commercial AZ31 Mg alloy could be refined to 1 μm after heavy deformation by equal channel angular pressing (ECAP) for 8 passes (effective strain ~9.2) at a temperature of 200 °C. Moreover, Mukai et al.16 have also demonstrated that the ductility in AZ31 and WE43 Mg alloys was significantly enhanced by the grain refinement. Recently, we have reported that the average grain size of a precipitation hardened Mg alloy (ZKX600) could be refined to 100 nm after SPD using high pressure torsion (HPT) by 1 rotation (360°) at room temperature. Furthermore, fully recrystallized ultrafine grained (UFG) structures which have not been achieved by other researchers were also obtained in the HPT processed material after subsequent rapid annealing17.On the other hand, Mg alloys can be deformed to ultra-high strains without cracking by severe plastic deformation (SPD) at relatively low or even ambient temperature18. In contrast, fully-recrystallized UFG ZKX600 alloy specimens exhibited both enhanced strength and ductility compared to those of the starting material having conventional coarse grained microstructure17. However, detailed strengthening and toughening mechanisms have not been clarified yet. Thus, the main purpose of this paper is to reveal the influence of grain size on the change of deformation mechanism in the ZKX600 alloy. We carefully observed the deformed microstructures of the specimens having two typical grain sizes and found that the dominant deformation mechanisms  have been significantly changed in the UFG specimen.It has been known that plastic instability happens in most UFG materials at early stages of tensile deformation because of highly enhanced yield strength and limitation of strain hardening in UFG structures, and thus it is difficult to manage both high strength and enough ductility in bulk UFG materials. For example, UFG Al and IF steel having sub-micrometer grain sizes exhibited quite early plastic instability, so that their uniform elongation is limited within a few percents1 phase, whose structure was a hexagonal structure as reported previously in various Mg-Zn based alloys19. Statistical length distribution of the rod-shaped precipitates is shown in Fig. As mentioned in the last section, the ZKX600 alloy is a typical precipitation hardened Mg alloy. The change of nano-precipitates during the present processing was investigated by transmission electron microscopy (TEM). Figure 1 phase was refined by HPT. The histogram of the length of the precipitates  scanning transmission electron microscopy (STEM) mode Fig. . Since ttes Fig.  indicateage Fig.  showed bage Fig. . The con17. Thus, in the present study, the specimen HPT processed by 360° (corresponding shear strain: γ = 23.55) was annealed at various temperatures for different holding periods, and then the specimens with recrystallized grain sizes ranging from sub-micrometer to several tens of micrometers were obtained. The EBSD inverse pole figure (IPF) maps and corresponding grain boundary (GB) maps of several representative specimens are displayed in Fig. It has been clarified by our previous study that saturated UFG microstructure could be realized after HPT by 360° rotationet al.21 have studied the texture evolution in a ZK60A Mg alloy during HPT and they found that a significant difference in the evolution of texture between the overall texture taken throughout the disc and the local texture at the disk edges up to at least 5 turns of HPT. That means, the selection of the measured area is critical for texture analysis in HPT processed Mg. In the present study, local areas corresponded to the center of the gauge part of the tensile specimens  of the 360° HPT processed specimen (d = 0.1 μm) reached 311 ± 3 MPa. However, premature fracture took place at a very limited total elongation (TE) of 1.2 ± 0.1% . The low ductility was primarily due to their very high YS and low work-hardening capability, which was caused by their very small grain sizes and high density of dislocations retained in the microstructure26. Interestingly, fully recrystallized UFG specimens could manage both high strength and large ductility. Particularly, the specimen with the finest recrystallized grain size (d = 0.77 μm) exhibited superior ductility (uniform elongation (UE)~20.5 ± 1.0%, TE~26.1 ± 1.4%), without sacrificing high ultimate tensile strength . With increasing the grain size, the strength decreased gradually, but the change in tensile elongations was not significant. For example, the UE and TE were maintained at 22.7 ± 1.8% and 28.0 ± 1.3%, respectively, in the specimen having a mean grain size of 6.11 μm. Strength and ductility decreased simultaneously by further increase of the grain size. The YS and TE decreased to 90 ± 5 MPa and 20.3 ± 2.0%, respectively, in the specimen having a coarse grain size of 23.3 μm. It is worth pointing out that, in addition to grain growth, coarsening of precipitates (β′1 phase) may take place during the long-time annealing treatment at high temperatures, which can also result in the decrease of the tensile strength.Nominal stress-strain curves of the as-deformed and the as-annealed specimens are displayed in Fig. Θ = dσ/dε, where σ and ε are the true stress and true strain, respectively), since uniform elongation in tensile tests is determined by the plastic instability condition . The Θ-ε curves and σ-ε curves of several representative specimens are plotted in Fig. Θ decreased quickly at very beginning of plastic deformation, followed by an increase of Θ upon further deformation, resulting in a V-shaped work-hardening curve at small strains (below 0.03). After that, the Θ value became higher than that of the coarse grained specimen (d = 23.3 μm), leading to relatively larger uniform elongation. As the grain size increased, the V-shaped valley became less and less obvious and eventually disappeared.It is well known that the large tensile ductility is often derived from the high work-hardening rate  and a coarse grained specimen (d = 23.3 μm) at various tensile strains were analyzed by a digital image correlation (DIC) technique29. As shown in Fig. It is considered that the V-shaped change of the work-hardening rate at small strains in the UFG specimens was related to the discontinuous yielding phenomena involving Lüders deformationd = 23.3 μm) were selected for detailed investigations on deformed microstructures. First, we focused on deformation twinning. Figure e = 0) was rather random. Besides, only a few {10–12} <10–11> twins (ftwin = 0.1%) were observed, which were considered to be introduced by mechanical polishing. At a tensile strain of 0.03, a fairly large number of lenticular-shaped deformation twins (ftwin = 8.8%) were formed. They were all {10–12} <10–11> twins, whose CRSS is significantly lower than other twinning systems in Mg alloys30. Since the {10–12} <10–11> twin rotates the lattice by 86°, it can effectively accommodate the extension strain along c-axis32. In addition, most of the twins appeared in blue or green colors in the partitioned EBSD-IPF maps  decreased with increasing tensile strain, supported such a tendency. Further deformation to a strain of 0.08, the area fraction of {10–12} <10–11> twins dramatically increased to 16.4%, and mutual intersection of twins were frequently observed. It is interesting that some grains were completely replaced by twins. However, at a strain of 0.15, which was almost twice as that of the previous stage (0.08), there was only a slight increase of the twined area (18.6%), indicating a saturation of deformation twinning around this strain level.A CG specimen  was used to distinguish the types of dislocations 34. Figure g) in Fig. g·b = 0” criterion, dislocations having <c> component are invisible in Fig. a> type dislocations should disappear in Fig. (a)) were observed. These dislocations were most likely basal dislocations having <a> component in Burgers vector. On the other hand, Fig. c> component in Burgers vector were still not activated. It should be noted that the activation of non-basal dislocations have been reported in some Ca-containing Mg alloys 36. However, those alloys were mostly simple solid solution Mg alloys. In contrast, the distribution of Ca in precipitation hardened Mg alloys were inhomogeneous. For example, in Mg-Zn based alloys, Ca precipitates out with Zn, forming nano-precipitates37. Thus, the role of Ca in the present quaternary alloy is somehow complicated and further investigations on this issue should be done in the future.The surface of the tensile specimen became very rough at higher strain levels and it was difficult to determine the activated slip systems through the EBSD assisted slip trace analysis. Instead, we carried out TEM observations of the CG specimen deformed to a tensile strain of 0.10. The “).Therefore, it is considered that the plastic deformation of the CG specimen was predominated by the {10–12} <10–11> twinning and {0001} <11–20> basal slip. The quick decrease of work-hardening rate was mainly due to the rapid growth and saturation of deformation twins, as indicated by the deformation microstructures observed Figs –7.7d = 0.98 μm), in the same way as those for the CG specimen  was totally free of twins, suggesting that the grain-refinement strengthening inhibited deformation twinning even during mechanical polishing. At a tensile strain of 0.04, only 0.3% of deformation twins were formed, which was significantly lower than that of the CG specimen (8.8%) at a similar tensile strain (e = 0.03). Again, these twins were exclusively {10–12} <10–11> twins. With the increase of tensile strain, the area fraction of twins increased gradually. Even at a strain of 0.14, however, the fraction of twins was only 1.5%, which was again far less than that in the CG specimen (18.6%) deformed to a similar strain.Identical area EBSD observations of deformation twinning was also conducted on the UFG specimen  exhibited very large room temperature tensile elongation of 0.47, which was mainly due to the cross-slip to non-basal planes induced by plastic compatibility. In contrast, Cepeda et al.39 reported reduced tensile ductility in the pure Mg when grain size was refined from 36 μm to 5 μm. Their slip trace analysis suggested a transition from the non-basal slips dominated deformation in the coarse grained (36 μm) specimen to the basal slip dominated deformation in the fine grained (5 μm) specimen, which was totally opposite to the result reported by Koike et al.38. Thus, we focused on investigating the active slip systems in the UFG specimen (d = 0.98 μm).Figure 20 zone axis by a minimum tilting. As indicated by the SAED pattern inserted in Fig. 38. Figure (a), from which profuse dislocations aligned roughly parallel to the basal trace (the broken line in (b)) were observed. These dislocations were most likely basal dislocations. Besides, some curved dislocation segments (indicated by red arrows) that were not parallel to the basal trace were also observed, indicating some non-basal slip systems were also activated. In order to know the Burgers vector of these dislocations, this grain was further observed from near the20 zone axis under two beam conditions. The operation vector (g) in Fig. a>. We also checked many other grains and they all exhibited similar dislocation configurations. Thus, we can conclude that the non-basal slip system having <c> component such as {11–22} second-order pyramidal slip was not activated at this strain level. The dislocation segments that were not parallel to the basal trace  were shown in Fig. c> component. These dislocations were further observed at higher magnification under a weak-beam dark field (WBDF) condition  operation40. The diffraction contrast (dislocation segments having bright contrast) in Fig. c> component were activated at this strain level in the UFG specimen. Therefore, we can conclude that deformation twinning was significantly suppressed throughout the entire deformation process in the UFG specimens, whereas the non-basal slip systems having <c> component Burgers vectors were activated to realize three-dimensional plastic deformation. The activation of the unusual slip systems is considered to contribute to the enhanced work-hardening rate in the UFG specimen, as is discussed in the next section.The UFG specimen was further observed at a tensile strain of 0.095 by TEM. Figure ion Fig.  through ), the relationship between the work-hardening behavior and the dominated deformation modes at various strain levels in the CG and UFG specimens is schematically revealed in Fig. 45. Afterward, the subsequent quick decrease of work-hardening rate is due to the deceleration and saturation of deformation twinning at the high strain level, as confirmed in Fig. a> component dominated the plastic deformation at the early stage. Subsequently, <c + a> dislocations were activated, which played a crucial role in strengthening the material, especially in producing deformation along the <c> axis. Additionally, activation of dislocations having totally different Burgers vectors would enhance interactions between dislocations, leading to an increase of work-hardening46, which was supported by the high density of tangled dislocations observed in Fig. Based on the systematic observations of deformation microstructures Figs –10, the 10et al.47 reported that a bulk specimen of pure Mg with a mean grain size of about 1 μm could be compressed to a large strain of 85% without fracture. Their observation of deformation microstructures and strain rate jump test suggested that intergranular deformation modes such as grain boundary sliding were responsible for the good formability of the UFG pure Mg. However, for the highly alloyed Mg-Zn-Zr-Ca alloy studied in the present study, the situation is greatly different. As shown in Fig. It should also be noted that, in addition to the activation of unusual slip systems, some other mechanisms such as grain boundary sliding are also expected to be important in UFG Mg, which could improve the ductility or formability of Mg. For example, Zeng c> component could be activated when the grain size was refined down to sub-micrometer scale. Consequently, synergetic improvement of strength and ductility was realized in the UFG specimen. The reason for the activation of unusual slip systems is still unclear. It is noteworthy, however, that UFG materials that can manage both high strength and large ductility have been recently found in particular alloys such as Cu-Al49 and high-Mn steels51, although it was considered that ultra grain refinement generally leads to the decrease of uniform elongation owing to early plastic instability. For example, in Cu-Al alloys having single-phased FCC structure with very low stacking fault energies, deformation twinning was found to be enhanced in fully recrystallized UFG microstructures49, which was totally opposite to the well-known grain size dependence of deformation twinning, i.e., deformation twinning becomes difficult to occur with decreasing the grain size. All those results suggest that there is an unknown mechanism to enhance unusual deformation modes in fully recrystallized UFG structures.It is an important and new finding that unusual slip systems having <d = 23.3 μm) was predominated by {10–12} <10–11> twinning and {0001} <11–20> basal slip, and the quick decrease of work-hardening rate at the later stage of plastic deformation was due to the deceleration and saturation of deformation twinning, (ii) deformation twinning was dramatically suppressed in the UFG specimen (d = 0.98 μm), while non-basal slip systems having <c> component Burgers vectors were activated successively. Particularly, the operation of the <c> component dislocations was responsible for the enhanced work-hardening rate. The present results showed a new insight that hexagonal Mg alloys can be very ductile by activating unusual deformation mechanisms (<c + a> dislocations) through realizing fully recrystallized UFG microstructures.In summary, the change of mechanical properties and deformation mechanisms in fully recrystallized UFG specimens were systematically investigated. We found that: (i) the plastic deformation of the coarse grained specimen  alloy produced by induction melting in Ar was used as the starting material. The as-cast alloy was provided for a multi-step thermo-mechanical processing, including solution treatment (ST), HPT to various rotation angles, and subsequent annealing treatment. The details of processing can be found in our previous publication4)2, 500 ml methanol and 100 ml 2-butoxy-ethanol at a voltage of 65 V and temperature approximately −50 °C.Microstructures of the specimens having various grain sizes were examined using scanning electron microscopy (SEM) and electron backscattering diffraction (EBSD). The specimens for SEM and EBSD analysis were mechanically polished using a fine grade sandpaper followed by a final polish using a colloidal silica suspension . EBSD results were analysed by TSL OIM software. Dislocation structures were observed by transmission electron microscopy (TEM). TEM specimens were firstly mechanical polished to 100 μm in thickness and then twin-jet electropolished using a solution of 5.3 g LiCl, 11.6 g Mg(ClO−4 s−1. The selected positions for microstructure observations and tensile tests as well as the detailed dimensions of the tensile specimen are shown in Fig. Mechanical properties of the specimens having various grain sizes were characterized by tensile test with an initial strain rate of 8.3 × 10Supplementary Information


It is well-established that angiotensin II exerts a dampening effect on the baroreflex within the nucleus tractus solitarii (NTS), the principal brainstem site for termination of baroreceptor afferents and which is densely populated with gamma-aminobutyric acid (GABA)ergic neurons and nerve terminals. The present study was designed to investigate whether local release of GABA is involved in the effects mediated by local angiotensin II within the NTS.In vivo microdialysis was used for measurement of extracellular glutamate and GABA levels and for infusion of angiotensin II within the NTS of conscious normotensive Wistar rats. The mean arterial pressure (MAP) and heart rate response to local infusion of angiotensin II were subsequently monitored with a pressure transducer under anesthesia. The angiotensin II type 1 receptor (AT1R) antagonist, candesartan, was used to assess whether responses were AT1R dependent and the nitric oxide (NO) synthase inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME), was used to assess the involvement of NO in the evoked responses by infusion of angiotensin II. The MAP and heart rate responses were monitored with a pressure transducer.L-NAME, demonstrating that the effect is not only AT1R dependent but also NO dependent. The pressor response to angiotensin II was reversed by co-infusion with the GABAA receptor antagonist, bicuculline. Local blockade of NO synthase decreased both, GABA and glutamate concentrations.Local infusion into the NTS of angiotensin II induced a significant to ninefold significantly increase in extracellular GABA levels; as well as MAP was increased by 15 mmHg. These responses were both abolished by co-infusion of either, the angiotensin II type 1 receptor antagonist, candesartan, or the NO synthase inhibitor, Our results suggest that the AT1R mediated hypertensive response to angiotensin II within the NTS in normotensive rats is GABA and NO dependent. Nitric oxide produced within the NTS tonically potentiates local GABA and glutamate release. The central nervous system network that regulates the level of sympathetic tone and, hence, blood pressure, is located in the brainstem and consists mainly of the rostral ventrolateral medulla (RVLM), the paraventricular nucleus (PVN) of the hypothalamus and the nucleus tractus solitarii (NTS) . It is wThe role of the brain renin–angiotensin system (RAS), and in particular of the angiotensin II type 1 receptor (AT1R), in the regulation of blood pressure and sympathetic tone is also well-established . Brain aA- and GABAB- receptors  in other brain area’s , and as 04 maintenance diet). The sodium content of the food was 2.500 mg/kg implying that the daily sodium intake was approximately 54 mg per day. All procedures used and described for animal experiments were carried out in accordance with the National and European guidelines for animal experimental research (2010/63/EU) and were approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel, Belgium (Project No. 18-213-1). All efforts were made to avoid animal suffering. To minimize the number of animals used, each animal was used for microdialysis sampling as well as for MAP measurement and a control/baseline period before – and a wash out period after compound administration in the same animal. The number of rats used for each experiment was based on power calculations, which were approved by the Ethical Committee. In accordance with the ARRIVE Guidelines  weighing 250–300 g at time of surgery. Animals acclimatized at least 1 week to the animal facility before surgery at constant temperature (22 ± 3°C), a relative humidity of 55% ( ± 10%), a 12 h light–dark cycle and with free access to water and food (Safe Aidelines  and the N(ω)-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma Aldrich, Co. . Ang II was used as an AT1R agonist, bicuculline as a GABAA receptor antagonist and L-NAME as an inhibitor of NO-synthase (NOS). Candesartan was used as a competitive-insurmountable AT1R antagonist . Infusion doses of Ang II, bicuculline, L-NAME and candesartan were selected based on previous studies , candesartan (0.5 and 1.5 ng μL−1h−1), Ang II (1 and 3 μg μL−1 h−1) + candesartan (0.5 ng μL−1 h−1), Ang II (3 μg μL−1 h−1) + candesartan (1.5 ng μL−1 h−1), Ang II (3 μg μL−1h−1) + bicuculline (4 μg μL−1 h−1), bicuculline (4 μg μL−1 h−1), Ang II (3 μg μL−1 h−1) + L-NAME (0.4 μg μL−1 h−1), L-NAME (0.4 μg μL−1 h−1)] were dissolved in modified Ringer’s solution  and perfused at a flow rate of 2 μL min−1 through the microdialysis probe.The treatment compounds [Ang II  in order to reestablish blood brain barrier integrity and preceed gliotic reactions. Based on several studies  microdiaDay 3: The same rats as used for microdialysis experiments were anesthetized prior to cannulation of the right carotid artery for continuous monitor the MAP and HR as described in section “Mean Arterial Pressure and Heart Rate Measurement.”On the first day of the experiment, all animals were anesthetized prior to surgery with ketamine/diazepam mixture (90/4.5 mg/kg) intraperitoneally and received 4 mg/kg ketoprofen subcutaneously to prevent post-operative pain and inflammation. A stainless steel microdialysis guide cannula  was stereotactically implanted 2 mm above the left NTS , as described previously .−1 with modified Ringer’s solution . On the second day, dialysis samples were collected from freely moving rats allowing us to measure neurotransmitters at many time points in each animal. Samples were taken every 20 min and each experiment lasted for 6 h. Each experiment started with the collection of intracerebral baseline samples (1–6) for 2 h, during which the perfusion fluid was composed of modified Ringer’s solution. Consecutively, treatment compounds dissolved in modified Ringer’s solution were perfused through the microdialysis probe from the 7th collection period onward until the 12th collection. Finally, samples (13–18) were taken under infusion of Ringer’s solution alone.After recovery from the stereotactical guide implantation, rats were single housed in experimental cages with free access to food and water and the microdialysis probe  was inserted into the guide-cannula. The microdialysis probe was connected by tubings  to a micro-injection pump (BAS MD-1001). The microdialysis probe was continuously perfused overnight at a flow rate of 1 μL minGlutamate and GABA concentrations were measured in the dialysates using respectively, reversed-phase narrowbore LC with gradient elution and fluorescence detection and reversed-phase microbore LC with isocratic elution and electrochemical detection, as described in detail in previous publications .On the third day of the experiment, animals were anesthetized by 4% sevuflurane gas in order to continuously record MAP and HR via cannulation of the right carotid artery using a pressure transducer connected to a monitor (Phillips IntelliVue MP50) as described previously . The rigAt the end of every experiment, rats were sacrificed by an overdose of pentobarbital . Removed rat brains were kept on 4% paraformaldehyde solution. Probe localization was histologically verified after brain slicing by a neutral red staining compared against an anatomic atlas  in orderpost hoc test. All MAP and HR measurements were also shown as percentage of the baseline levels ± SEM. To determine intragroup differences, data were analyzed applying a Friedman test followed by the Dunnett’s multiple comparison test.Statistical analyses were performed using GraphPad Prism 6.01  with the α level chosen at 0.05. Data are expressed as mean ± standard error of the mean (SEM). The mean values of the basal microdialysis samples as well as of the baseline MAP and HR obtained before compound administration were considered as the 100% baseline value for each animal. Results for the neurotransmitters, glutamate and GABA, were expressed as percentages of the baseline levels ± SEM. Subsequently, for the microdialysis results, an area under the curve (AUC) analysis was performed to determine if there was a difference in GABA concentrations. AUC values, expressed in arbitrary units, were compared by a Friedman test with Dunnett’s multiple comparison −1 h−1) within the NTS on extracellular glutamate (n = 7 (1 μg μL−1 h−1); n = 6 (3 μg μL−1 h−1); −1 h−1 Ang II within the NTS  of the AT1R antagonist candesartan alone and during co-infusion with the two doses of Ang II were tested (−1 h−1) of candesartan alone (data not shown). The previously observed trend to an increase in GABA during infusion of 1 μg μL−1 h−1 Ang II was not seen during co-infusion with 0.5 ng μL−1 h−1 candesartan . Glutamate concentrations were not altered by co-infusion of 0.5 ng μL−1 h−1 candesartan with 1 μg μL−1 h−1 Ang II  significantly increased the GABA levels , was tested alone and during co-infusion with 3 μg μL−1 h−1 Ang II (L-NAME (0.4 μg μL−1 h−1) alone significantly (p < 0.05–0.01 compared to vehicle) reduced baseline glutamate levels  reduced but not different from levels during L-NAME (0.4 μg μL−1 h−1) alone  decreased the GABA levels and reversed the Ang II evoked increase in GABA within the NTS  or 3 μg μL−1 h−1 (n = 6) of Ang II compared to vehicle . There were no significant differences in MAP nor HR between vehicle and co-infusion of 1.5 ng μL−1 h−1 candesartan with 3 μg μL−1 h−1 Ang II , alone within the NTS did not modify the baseline MAP nor HR (data not shown). Co-infusion of 4 μg μL−1 h−1 bicuculline with 3 μg μL−1 h−1 Ang II significantly decreased the MAP at time point 110 min  alone within the NTS did not modify baseline MAP (n = 5) nor HR (n = 4). Co-infusion of L-NAME abolished the MAP increase induced by 3 μg μL−1 h−1 Ang II neurons within the NTS resulting in an increase in GABA release. An alternative possibility is that the Ang II mediated release of GABA from local inhibitory GABAergic interneurons within the NTS occurs indirectly and that Ang II stimulates the release of NO which is then responsible for increased GABA release. Our observation that the AT1R mediated increase in GABA within the NTS is abolished by co-infusion of the NOS inhibitor h groups .in situ ‘working heart-brainstem preparation’ (N(ω)-methyl-L-arginine acetate (L-NMMA), another NOS inhibitor, was shown by Ang II can indeed stimulate NO release , and an aration’ . MoreoveL-NAME alone tended to decrease GABA concentrations within the NTS suggesting the presence of an endogenous tone stimulating GABA release, in agreement with earlier suggestions based on gene transfer studies in conscious rats that eNOS is constitutively active within the NTS in conscious rats as a factor regulating baroreceptor reflex gain  receptor stimulation, can increase glutamate release within the NTS , with a subsequent local production of NO, which enhances GABA release probably from NTS interneurons. We speculate that this release of GABA within the NTS inhibits glutamatergic neurons projecting to the CVLM, thereby reducing the activation of the CVLM inhibitory GABAergic nerves with subsequent disinhibition of the sympathetic driving glutamatergic neurons in the RVLM. Our results further suggest that NO produced within the NTS tonically potentiates local GABA and glutamate release, and may therefore also be an important modulator of the central blood pressure regulation.This study was carried out in accordance with the recommendations of “National and European guidelines for animal experimental research, Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel, Belgium.” The protocol was approved by the “Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel, Belgium.”LL performed the research and wrote the research manuscript. IS and AD designed the research study and gave critical insights in the research manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A Bayesian approach was used to calculate N2O emission factors (EFs) and their associated uncertainties from flux chamber measurements made after the application of nitrogen fertilisers, in the form of ammonium nitrate (AN), urea (Ur) and urea treated with Agrotain® urease inhibitor (UI) at four grassland sites in the UK. The comparison between the cumulative fluxes estimated using the Bayesian and linear interpolation methods were broadly similar (R2 = 0.79); however, the Bayesian method was capable of providing realistic uncertainties when a limited number of data points is available. The study reports mean EF values  of 0.60 ± 0.63, 0.29 ± 0.22 and 0.26 ± 0.17% of applied N emitted as N2O for the AN, Ur and UI treatments, respectively. There was no significant difference between N2O emissions from the Ur and UI treatments. In the case of the automatic chamber data collected at one site in this study, the data did not fit the log-normal model, implying that more complex models may be needed, particularly for measurement data with high temporal resolution.Trapezoidal integration by linear interpolation of data points is by far the most commonly used method of cumulative flux calculations of nitrous oxide (N •2O flux estimates after fertiliser application.Bayesian statistics provided realistic uncertainties in cumulative N•2O were observed between urea fertiliser and urea treated with urease inhibitor.No significant difference in emissions of N•2O after the application of ammonium nitrate fertilisers is shown to be significantly higher than those of urea based fertilisers.Emissions of N Due to the unpredictable spatial and temporal variability in N2O fluxes from agricultural soils, estimates of emissions of N2O after fertiliser events have large associated uncertainties  integration between points is by far the most commonly used method of cumulative flux estimation. However, this method does not provide meaningful uncertainties in estimates. To better understand the results of Nstimates . Through2O emissions, it has been suggested that the IPCC Tier 1 method  thiophosphoric triamide, Koch, KS, USA) on intensively managed grassland silage crops at four sites in the UK, and compare this with the two most commonly used synthetic nitrogen fertilisers in the UK: ammonium nitrate  and urea. The results will then be used to analyse the feasibility of a Tier 2 EF method for regional and national N2O emission inventories.This study aims to use a Bayesian method for estimating cumulative fluxes  to quant22.1Four trials were conducted at intensively managed grassland sites in the UK, during the growing seasons in 2016 and 2017 .Table 1CThe trials were carried out at Easter Bush farm estate  , Henfaes−1) The UJ field had predominantly been used for silage harvest with occasional grazing during winter. The HF and NW sites were managed similarly, predominantly used for silage harvest with occasional grazing during winter months  and Upper Joiner (UJ) field sites. The EB field had historically been used to graze sheep , positioned randomly to mitigate bias resulting from spatial variability of soil properties. The plot layout varied in the 2017 UJ trial, for which plots were arranged in a square grid, each measuring 20 m by 20 m with no spacing between them. Applications of nitrogen fertilisers in the form of ammonium nitrate (AN), urea (Ur), and urea treated with the Agrotain® urease inhibitor (UI) were applied to the plots  two or three times per site, each application was replicated on four plots with an additional four control plots to which no Nr fertiliser was applied  plastic pipe of 38 cm inner diameter (ID) and 22 cm height fitted with sealed lid and a flange at the base. The chambers were placed onto a plastic flanged collar that had been inserted several centimeters into the soil (on average 5 cm) to form a seal in the soil. A layer of draught sealant material held in place by four strong gripping clips formed an airtight seal between the chamber and the collar for the duration of the flux measurement. Chambers were closed for 60 min, during which four gas samples were collected via a syringe and a three-way tap fitted to the lid, at t = 0, 20, 40 and 60 min. Gas samples were stored in 20 ml glass vials, which were flushed with 100 ml of air from the syringe using a double needle. Samples were analysed using gas chromatography . At the EB and UJ sites the manual static chamber measurements were carried out daily for two weeks after fertilisation, then every second day for a further two to four weeks, with measurements only made only on working days (Monday to Friday) between 09:00 and 15:00 GMT.At all sites, measurements of Nt = 0, 20 and 40 min. Gas samples were stored in pre-evacuated 20 ml glass vial, which were flushed with 50 ml of air from the syringe using a double needle. Samples were analysed for N2O concentration using a Perkin Elmer 580 Gas Chromatograph (linked to a TurboMatrix 110 Headspace Autosampler)  were inserted, so that 15–20 cm of the chamber height remained above the soil surface. On each sampling occasion, lids were placed on the chambers and remained in place for 40 min with three gas samples collected via syringe, at sampler) . At the 2O Analyser, Los Gatos Research Inc. San Jose, CA, USA). Chamber bases were inserted into the soil and the chambers (50 × 50 × 20 cm) attached to the bases at surface height to ensure an air tight seal. Closing and opening of the chambers was controlled by pneumatic actuators. The chambers closed for a 30 min measurement period (four times a day), during which the chamber-headspace was sampled via a sampling port at a rate of 1 l min−1 at a frequency of 0.1 Hz.Further measurements were made at the HF site using an automatic chamber approach (via an Isotopic N−2 s−1) dC/dt is the rate of change in the concentration in time in nmol mol−1 s−1 estimated by linear regression, ρ is the density of air in mol m−3, V is the volume of the chamber in m3 and A is the ground area enclosed by the chamber in m2.Fluxes were calculated as:2.32O fluxes, F, are typically log-normally distributed in space, the probability density is given by:μlog and σlog are the location and scale parameters, equivalent to the mean and standard deviation of the log-transformed variate. The mean of the distribution is given by:Cumulative fluxes over the experimental periods (30 days) were calculated using a Bayesian approach, taking into account the log-normal distribution of spatial samples and the lognormal peak-and-decay pattern in time . Based o2O flux is expected to rise to a peak, then decay exponentially, and this basic pattern is reproduced by all process-based models  method with Gibbs sampling , a cumulative background flux was estimated using the mean of the fluxes measured from the control plots during each event. This cumulative background estimate was then subtracted from the cumulative fluxes estimated for each treatment. The reported EFs in this study take background fluxes into account when reporting final values , but overall the comparison was good. Taking background emissions into account, EFs ranged from slightly negative values  to a maximum of 1.92% of the N applied.Cumulative fluxes calculated using the linear and Bayesian methods were broadly comparable across the different sites. The direct comparison of the two interpolation methods shows that in this study the Bayesian method predicted slightly larger fluxes than the linear method (slope = 1.04) and there is some disagreement in larger EF estimates , not breaching the 0.5% mark in any experiment . Based on the posterior distribution provided by the Bayesian method, EFs associated with AN treatments were found to be larger than those of the urea treatments 55% of the time and that the EFs associated with UI were larger than Ur 60% of the time; however, the magnitude of these differences was inconsistent and not statistically significant.2O than equivalent applications of urea . However, it is very difficult to accurately predict the microbial interactions which affect Neriments . Correlaeriments .2O over several hours that could be greater than cumulative emissions for several weeks afterwards, or alternatively, nothing at all under what appears to be similar conditions , as demonstrated by the contrast of AN and urea treatments . With a A comparison of the Bayesian and trapezoidal linear interpolation methods show that they did not differ systematically, but the former provides rigorous uncertainties when a limited number of data points is available. In this respect, the Bayesian method has been a success, as large uncertainties are provided when data varies significantly in time and space, something which was not possible to assess when using traditional trapezoidal linear interpolation method. However, care should be taken when using the Bayesian method when flux measurements are more temporally defined (i.e. auto-chambers and eddy covariance), as the log-normal fit becomes too constrained and uncertainty estimates are no longer valid.52O for the AN, Ur and UI treatments, respectively. The study found that EFs associated with AN were more likely to be larger than those of the urea-based treatments, but there was no significant difference in overall emissions of N2O between the Ur and the UI treatments. The Bayesian method used in this study successfully provided uncertainty values in cumulative fluxes of N2O that traditional trapezoidal linear interpolation methods could not. In its current form, the method is limited to cases where the emissions show a peak and decline following fertilisation, as expected from first principles.A Bayesian approach was used to calculate EFs and their associated uncertainties from the application of nitrogen fertilisers in the form of ammonium nitrate (AN), urea (Ur) and urea treated with Agrotain® urease inhibitor (UI) at four grassland sites in the UK. The study reports that the EFs observed after 11 separate events followed a variable log-normal distribution, with mean reported values of 0.60 ± 0.63, 0.29 ± 0.22 and 0.26 ± 0.17% of applied N emitted as N


We also observe a distinct conductance ‘jump’ at the end of the sliding process, which is further evidence that quantum interference effects dominate charge transport in these single-QD junctions. This conductance ‘jump’ is also confirmed by our theoretical calculations utilizing density functional theory combined with quantum transport theory. Our measurements and theory create a pathway to exploit quantum interference effects in quantum-controlled perovskite materials.The studies of quantum interference effects through bulk perovskite materials at the Ångstrom scale still remain as a major challenge. Herein, we provide the observation of room-temperature quantum interference effects in metal halide perovskite quantum dots (QDs) using the mechanically controllable break junction technique. Single-QD conductance measurements reveal that there are multiple conductance peaks for the CH Quantum interference effects remain elusive in halide perovskite materials. Here Zheng et al. reveal the atomic origin of the conductance features in the single perovskite quantum dot junctions, and present direct evidence of the room-temperature quantum interference effects. Previous experimental and theoretical investigations of room-temperature QI effects have mainly focused on organic molecular wires, including π-conducting wires3, σ-conducting wires4, and even π-stacked dimers5, but the exploitation of room-temperature QI effects in electron transport through Ångstrom-scale inorganic systems still remains unexplored. The unique quantum yields and high carrier mobility of perovskite-based electronic materials offer a platform for us to translate knowledge of their macroscopic charge transport into the quantum effects at the nanoscale.Quantum interference (QI) effects, originating from de Broglie waves of electrons traversing different pathways through nanoscale junctions, underpin the conceptual designs of molecular devices such as QI based field-effect transistors6, photodetector7, and solar cells9. Although there are many experimental investigations of charge transport through bulk perovskite materials, including thin films10, nanocrystals11, and single crystals12, investigations at the nanoscale, to reveal QI effects in their room-temperature transport properties remain as a major experimental challenge. The extensions of single-molecule charge transport measurements from conjugated molecular families13 to molecular assemblies15, clusters16, and the recently developed Au-halogen interfacial engineering17 offer an opportunity to gain an insight into microscopic charge transport through Ångstrom-scale perovskite materials.Perovskite materials attract extraordinary attention in applications of the light-emitting diode3 and MAPbBr2.15Cl0.85 (MA = CH3NH3+) QDs, while MAPbBr2.15I0.85, MAPbCl3 and MAPbI3 QDs show no significant conductance features. The displacement distributions also match well with the lattice constant of QDs, suggesting that the multiple conductance features are derived from the sliding of gold electrodes through different lattice sites of the QD via Au-halogen coupling. A distinct conductance ‘jump’ is also observed at the end of the sliding process, which is further evidence that QI effects dominate charge transport in the single-QD junctions.To understand how their macroscopic charge transport properties lead to quantum effects at the nanoscale, here we report the observation of room-temperature QI effects in metal halide perovskite quantum dots (QDs) at the Ångstrom scale using the mechanically controllable break junction (MCBJ) technique combined with quantum transport theory and calculations. Multiple distinguishable conductance peaks can be observed in the MAPbBr20, if the coupling between molecule and electrode is weak, the effect of QI on transport properties could be predicted by examining the Green’s function G(EF)of the isolated molecule. The transmission amplitude of an electron with energy EF from site i to j is proportional to nth molecular orbital (MO) on site i and εn is the corresponding MO energy level. Taking only the HOMO and LUMO into consideration and assuming that EF is located in the midgap of HOMO and LUMO, this equation could be further written as aH and aL have opposite signs, while destructive quantum interference (DQI), corresponding to a low value of aH and aL have the same sign. As an example of this sign dependence, if the electrodes make contact with the left and right ends of the perovskite cluster in Fig. aL) is negative. On the other hand, the HOMO has a positive amplitude on the left and a positive amplitude on the right, hence the product (aH) is positive. Therefore, CQI is expected. Hence when electrodes are attached to the perovskite junctions with ‘long’ or ‘short’ sites in Fig. The tunneling transport through QDs or molecules is mediated by electrons whose energy lies within the energy gap between the highest occupied molecular orbital (HOMO) and lowest occupied molecular orbital (LUMO), thus the inter-orbital QI can be understood qualitatively by inspecting the signs of the HOMO and LUMO at the points of contact between the molecule and electrode Fig. . As mentL = 2, 3, ……8)  should lie above the trend defined by the tunneling decay equation G~eβL−. The transmission function for this simple model  are synthesized with oleic acid and octylamine as ligands to enhance colloidal stability and suppress QD aggregation effects  in the four octahedra central positions. Single-QD conductance measurements of MAPbBr3 are carried out using the MCBJ technique in a solution containing 0.365 mg mL−1 QDs with 1, 3, 5-trimethylbenzene (TMB) as a solvent , while three distinguished conductance plateaus and ‘jump plateaus’ appear in the traces of MAPbBr3. To reveal the source of the conductance plateaus, we also carry out the MCBJ measurements of all ligands and ingredients used in the synthesis of the QDs in the solvent γ-butyrolactone, including oleic acid, octylamine, PbBr2, PbCl2, MACl, and MABr. The obvious conductance plateau can be observed in PbBr2, while no clear conductance signal can be observed in other ligands and raw materials, suggesting that the conductance signal may come from Au–Br interaction and the other ligands cannot form the single-QD junction  conductance-displacement histogram is plotted in Fig. 2.15Cl0.85, MAPbBr2.15I0.85, MAPbCl3 and MAPbI3 QDs. Figure 2.15Cl0.85, multiple conductance features are also observed, which are similar to those of MAPbBr3. The one-dimensional (1D) conductance histograms . As shown in Fig. −1 for MAPbBr3 and MAPbBr2.15Cl0.85, which confirms the formation of Au–Br bond28,  are calculated by combining the DFT package SIESTA29 with the quantum transport code Gollum30 (see Method for further details). The MAPbBr3 neutral charge clusters (1Pb (MA4PbBr6), 8Pb (MA20Pb8Br36), 12Pb (MA28Pb12Br52), 16Pb (MA36Pb16Br68)) are built with the same method as the literature31  and further to 3.91 eV (for 1Pb) due to the stronger quantum confinement effect. After the rupture of the gold wire, the initial gap width is known to be a snap-back distance of about 5 Å. Since this corresponds to the distance between two neighboring Br atoms (around 5 Å), these two Br atoms are most likely to be connected to the gold atoms at the beginning in this sliding process. As for the sliding direction, the gold electrode could slide from the top of one Br atom to its adjacent Br atom along the horizontal and diagonal directions , 8Pb MAPb8Br36, 3 cluster is connected to two gold electrodes through two Br atoms as shown in Fig. L. Therefore, we fit the room-temperature conductance (EF = −1.0 eV) to an exponential function, which led to an attenuation factor of β = 0.54 Å−1 was obtained, which is consistent with our measured value of 0.57 Å−1, as shown in Fig. 3), 16Pb and 18Pb MAPbBr3 clusters , and the latter connectivity is less likely to appear in the experiments due to the higher energy barrier.In the current study, the fully relaxed 12Pb MAPbBr3 and MAPbI3 QDs. Our results show that the three halide perovskite QDs possess similar charge transport features  are also considered in our calculations. As shown in Supplementary Figs. The influence of ligand (oleic acid and octylamine) on the transmission functions is investigated by considering the ligands staying close to the cluster or bridging the gold electrode and perovskite cluster  and 0.16 mmol MABr  is dissolved in 5 mL DMF. 0.5 mL oleic acid and 20 μL n-octylamine are added to obtain a stable precursor solution. The 1 mL precursor solution is rapidly injected into 5 mL toluene under the stirring with 800 rpm. In the stirring process, strong green PL emission from MAPbBr3 QDs can be observed under normal room light without using additional excitation light source. Then the precursor solution is centrifugated at 5000 or 10,000 rpm for 10 min to discard the precipitates.Perovskite QDs in this paper are synthesized according to published papers2 blowing. Transmission electron microscopy  with fast operation at 200 kV is employed to obtain TEM or HRTEM images before damaging perovskite QDs.Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM): a drop of diluted QDs solution is spread onto an ultrathin carbon film-coated copper grid and is further dried by gentle N−1 QDs at room temperature22. During the process of MCBJ measurement, the substrate is first fixed by two counter supports at both sides, and then a pushing rod driven by a stepping motor and a piezo stack is employed to bend the substrate in the middle, resulting in the breakage of the notched gold wire at the horizontal direction to form a nano-gap. The fractured gold wire will capture a single perovskite QD with halogen anchors and forms the Au-QD-Au junction. Owing to the elasticity of the stainless-steel substrate, the gold wire connects again during the returning process of the pushing rod. After repeating this process thousands of times, the individual conductance-distance traces of the single-QD junctions can be collected and further analyze the most probable conductance of the junctions.The single-molecule conductance measurements of perovskite QDs are carried out by using the MCBJ technique in a solution containing 0.365 mg mL29 geometrical optimizations were carried out until all the forces were less than 0.05 eV Å−1. A generalized gradient approximation functional (GGA), a double-ζ basis for Au, a double-ζ polarized basis for other elements and a real grid cutoff energy of 150 Ry were employed30. A scalar relativistic norm-conserving pseudopotential is used to describe Pb. To compute their electrical conductance, the molecules are each placed between pyramidal gold electrodes. After relaxation, the optimized separation between contact halogen atoms  and apex gold atom was found to be 2.66 Å, 2.76 Å and 2.88 Å, respectively. From the converged DFT calculation of each structure, the mean field Hamiltonian and overlap matrix are extracted, which are utilized to calculate the transmission coefficient T(E) using the Gollum code 30, via the expressionUsing the DFT code SIESTA L,R describes the level broadening due to the interaction between left (right) electrodes and the scattering region. S and H are the Hamiltonian and overlap matrix, respectively. The room-temperature conductance is obtained by the following formula:G0 = 2e2h−1 is the conductance quantum; h is the Planck’s constant; e is the charge of a proton; EF is the Fermi energy.In this equation, Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


More than 20% of the world’s agricultural land is affected by salinity, resulting in multibillion-dollar penalties and jeopardising food security. While the recent progress in molecular technologies has significantly advanced plant breeding for salinity stress tolerance, accurate plant phenotyping remains a bottleneck of many breeding programs. We have recently shown the existence of a strong causal link between salinity and oxidative stress tolerance in cereals (wheat and barley). Using the microelectrode ion flux estimation (MIFE) method, we have also found a major QTL conferring ROS control of ion flux in roots that coincided with the major QTL for the overall salinity stress tolerance. These findings open new (previously unexplored) prospects of improving salinity tolerance by pyramiding this trait alongside with other  mechanisms.2O2-triggered loss of root cell viability was observed, with salt sensitive varieties showing significantly more damage to root cells. In the root growth assays, relative root length (RRL) was measured in plants grown in paper rolls under different H2O2 concentrations. The biggest difference in RRL between contrasting varieties was observed for 1 mM H2O2 treatment. Under these conditions, a significant negative correlation in the reduction in RRL and the overall salinity tolerance is reported.In this work, two high-throughput phenotyping methods—viability assay and root growth assay—were tested and assessed as a viable alternative to the  MIFE method using barley as a check species. In viability staining experiments, a dose-dependent HThese findings offer plant breeders a convenient high throughput method to screen germplasm for oxidative stress tolerance, targeting root-based genes regulating ion homeostasis and thus conferring salinity stress tolerance in barley .The online version of this article (10.1186/s13007-019-0397-9) contains supplementary material, which is available to authorized users. Both global climate change and unsustainable agricultural practices resulted in significant soil salinization thus reducing crop yields , 2. UntiOne of constraints imposed by salinity stress on plants is an excessive production and accumulation of reactive oxygen species (ROS), causing oxidative stress. This results in a major perturbation to cellular ionic homeostasis  and, in + retention and lower Ca2+ uptake when challenged with H2O2 [microelectrode ion flux estimation (MIFE) technique. We have then applied this newly developed cell-based phenotyping MIFE platform to DH (double haploid) barley lines, revealing a major QTL for the above flux traits . These findings open exciting prospects for plant breeders to screen germplasm for oxidative stress tolerance, targeting root-based genes regulating ion homeostasis and thus conferring salinity stress tolerance. The bottleneck in application of this technique in breeding programs is a currently low throughput capacity and technical complications of using the MIFE method.Recently we have shown that roots of salt-tolerant barley and wheat varieties possessed greater Kith H2O2 . These iThe MIFE technique works as a non-invasive tool to monitor kinetics of net ion transport (uptake or release) across cellular membranes by using ion-selective microelectrodes . This isHordeum vulgare). The biological rationale behind these approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers programmed cell death [In this work, we attempted to seek much simpler alternative phenotyping methods that can be used as a proxy to screen cereal plants for oxidative stress tolerance. To do so, we developed and compared two high-throughput assays (a viability assay and a root growth assay) for oxidative stress screening of a representative cereal crop: barley (ll death –18 and rll death  and is aHordeum vulgare L. and one H. vulgare ssp. Spontaneum) genotypes were used in this study. All seeds were obtained from the Australian Winter Cereal Collection  in darkness at 24 ± 1 °C for another 3 days.Eleven barley  with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-pressure mercury lamp  was used to examine stained root segments. The excitation and emission wavelengths for FDA and PI were 450–495 nm and 495–570 nm respectively. Photographs were taken by a digital camera . Images were acquired and processed by LAS V3.8 software (Leica Microsystems). The exposure features of the camera were set to constant values  in each experiment, allowing direct comparison of various genotypes. For untreated roots, the exposure time was 591 ms; for H2O2-treated roots it was increased to 1.9 s. The overview of the experimental protocol for viability assay by the FDA - PI double staining method can be found in Additional file Viability assessment of barley root cells was performed using a double staining method that included fluorescein diacetate  and propidium iodide  . BrieflyImageJ software was used to quantify red fluorescence intensity that is indicative of the proportion of dead cells [ImageJ and colour channel of the image were split into green, red and blue. The “polygon selections” from the tool bar of ImageJ was used to select an area from root elongation (0.3 mm from root tip) and/or mature zone (anywhere of the cut mature root segments) from the image with red channel. The measurement was conducted by going to “Analyze” option at the main menu, followed by the “Measure” option. The results will be shown in a pop-up window containing the size of the measuring area chosen, the minimum/maximum/mean value of the red fluorescence intensity. Images of H2O2-treated roots were normalised using control (untreated) roots as a background. The red fluorescence intensity  was recorded as removal of intensity in untreated roots from H2O2-treated root in arbitrary units.The ad cells . The acq2O2 ranging between 0 and 10 mM . The relative root lengths (RRL) were estimated as percentage of root lengths to controls of the respective genotypes.The length of the longest seminal root was measured form 4-days old barley seedlings using a ruler after 3 days of treatments with various concentrations of HThe damage index was used as a measure of the overall salt stress tolerance. This index is based on the extent of leaf necrosis and chlorosis in plants exposed to prolonged (5–6 weeks) salinity and quantified on 0–10 scale  –26. Beint test at P < 0.05 level.Statistical significance of mean values was determined by the standard Student’s 2O2 on cell viability. The concentrations of H2O2 used was from 0.3 to 10 mM. Both 1 day-  . Accordingly, 10 mM H2O2 with 3 days treatment was chosen as the optimum experimental treatment for viability staining assays on contrasting barley varieties.The quantitative analysis of the red fluorescence intensity was implemented in order to translate images into numerical values Fig. . Mild ro2O2 in all barley varieties and both root zones, and damage in the elongation zone was more severe than in the mature zone  were employed to explore the extent of root damage upon oxidative stress by the means of viability staining of both elongation and mature root zones. A visual assessment showed a clear root damage upon 3 days-exposure to 10 mM HThe quantitative analysis of the fluorescence intensity revealed that salt sensitive varieties showed stronger red fluorescence signal in the root elongation zone than tolerant ones Fig. a, indica2O2-induced K+ and Ca2+ fluxes were about the same for barley and wheat [The results in this section were consistent with our previous findings by using MIFE technique , which end wheat , we have2O2 for 3 days that would include staining the root tips with FDA and PI, and then quantifying intensity of the red fluorescence signal (dead cells) from the root elongation zone. This protocol is simpler and quicker than MIFE assessment and requires only a few minutes of measurements per sample, making this assay compliant with the requirements for high throughput assays.Based on these findings, we can conclude that plant oxidative and salinity stress tolerance in barley may be quantified by the viability staining of roots treated with 10 mM H2O2) treatment in preliminary experiments. After 3 days of H2O2 treatment, root length declined in both the varieties for any given concentration tested , and salt tolerant variety TX 9425 grew better  than salt sensitive variety Naso Nijo at each treatment used  occurring from 1 to 10 mM H2O2 treatments between the contrasting varieties , and thus may be not easily applicable by all the breeders. This has prompted us to go along another avenue by testing root growth assays. Two contrasting barley varieties, TX 9425  and Naso Nijo  were used for standardizing concentration of ROS  of all the barley varieties reduced rapidly, ranging from the lowest 22.7% ± 0.3 (in the variety Unicorn) to the highest 63.2% ± 2 (in SYR01)  between RRL and the overall salinity tolerance was observed  Fig. b. The RRved Fig. c, indica2O2 is one of the major ROS produced in plant tissues under stress conditions that leads to oxidative damage. The effect of this stable oxidant on plant cell viability and root growth was investigated in this study. Both parameters decreased in a dose- and/or time-dependent manner upon H2O2 exposure  levels of cytosolic free Ca2+ [2O2-induced cytosolic Ca2+ disequilibrium may be another contributing factor to the observed loss of cell viability and reported decrease in the relative root length in this study , we observed less severe root damage in the elongation zone in salt tolerant varieties  to stress defence mechanisms [+-ATPases activity in many species [It was argued that the ROS-induced self-amplification mechanism between Cagnalling , 39, 40.gnalling , this maeriments . Under lchanisms , which mchanisms . This is species –45, and  species .+ efflux in response to H2O2 [2O2 exposure  channels in Vicia Faba guard cells [+ back leak into cytosol from the vacuole pool. This observation was later extended to cell types isolated from Arabidopsis shoot and root tissues [2+ influx in sensitive varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+ concentration thus inducing a strong K+ leak from the vacuole, to compensate for the cytosolic K+ loss from ROS-activated GORK channel. This process will be attenuated in the salt tolerant varieties, that have lower H2O2-induced Ca2+ uptake. As a result, 3 days after the stress onset, the amount of K+ in the cytosol in mature root zone may be not different between contrasting varieties, explaining the lack of difference in viability staining.In the mature root zone, the salt-sensitive varieties possessed a higher transient K to H2O2 ; yet no re Figs. b, 4b. Thpartment . Increasrd cells , mediati tissues , as well tissues . Thus, tA rapid and revolutionary progress in plant molecular breeding has been witnessed since the development of molecular markers in the 1980s . At the 2O2 used and acquiring the damage index scores of these plants.In this work, this method was only tested in barley used as a check species. However, given the fact that salt-induced increase in ROS production in roots is a general biological phenomenon –54, we e2O2-treated root elongation zone, or by measuring roots lengths after the same treatment with the former. However, the latter method is low/no-cost and easy-to-implement thus may be recommended as an alternative phenotyping method in future screening programs.Our results show that oxidative and salinity stress tolerance phenotyping in barley may be conducted by staining with FDA and PI and then quantifying the intensity of red fluorescence signal from HAdditional file 1: Fig. S1. Flowchart of viability staining and fluorescence image acquisition. a Preparation of root sample for viability staining. (From left to right panel) Surfaced sterilized seeds were germinated in a large Petri dish with wet filter paper for 1 day. Uniformly germinated seeds were then chosen and placed in paper rolls before placing it in a beaker with growth media for another 3 days. Isolated root segments were placed in a micro Petri dish containing 5 µg/ml FDA for 5 min and then transferred to another micro Petri dish containing 3 µg/ml PI for 10 min; Stained root segments were washed with distilled water and positioned on a glass slide and covered with a cover slip. b The prepared slide was placed on a fluorescent microscope mechanical stage under the fluorescent light and root fluorescent image was acquired by the LAS V3.8 software.