Epichloë endophytes have been shown to increase tolerance to biotic and abiotic stresses in many cool-season grasses. We investigated the impact of endophyte infection of Festuca sinensis, on root metabolic activity, photosynthetic pigments, leaf relative water content (RWC) and soluble carbohydrates in a field experiment carried out during chilling and irrigation with Ca(NO3)2. A highly significant (P < 0.001) correlation for Epichloë endophytes was observed for root metabolic activity. Ca(NO3)2 affected very significantly root metabolic activity and total chlorophyll (P < 0.001). Low temperature led to highly significant (P < 0.001) reductions in root metabolic activity, RWC, total chlorophyll, chlorophyll a/b ratio, and carotenoid contents. In addition, the fructose concentrations of shoots were greater on the 14th day than on the 28th day and before treatment, whilst the glucose concentration of roots was much higher on the 28th day than before and after 14 days treatment. Moreover, our results indicated that the addition of calcium nitrate contributed to higher levels of total chlorophylls, soluble sugars, sucrose, fructose or glucose in the shoots and roots in both E+ and E- plants during long periods of chilling. These results suggest that Epichloë endophyte infection and/or exogenous calcium nitrate can confer better tolerance to cold stress. Nayyar, Bains & Kumar, 2005). There is considerable evidence that exposure to low temperature causes a variety of morphological, physiological and biochemical responses, including declines in chlorophyll content , root metabolic activity , and accumulation of soluble carbohydrates , such as drought , temperature , oxidate , salt , and enhancing sugar contents , salt  and meta2), salt . Exogeno2), salt , increascontents . Cold stnditions .Yin et al., 2014). Epichloë endophytes . In addition, other symbioses are reported to affect photosynthetic pigment degradation, leaf relative water content, soluble sugars and other physiological markers for chilling stress . Cold winter conditions can lead to damage or death of grasses. However, despite this survival of F. sinensis it is still stressed under long-term cold exposure in grasslands of northwestern China.of China . This grnd seeds . SeveralEpichloë endophytes in stimulation of root metabolic activity, photosynthetic pigment accumulation, RWC and soluble carbohydrates, and how these may improve F. sinensis survival under cold conditions in the field.A previous study has suggested that cold stress negatively affects nitrogen uptake by plants . The aimFestuca sinensis seeds (E+) that were naturally infected with the endophyte were collected in September 2012 from Xiahe county, Gansu Provence in China . To obtain endophyte free (E- ) seeds, Epichloë endophyte infected seeds (E+) were treated with 300 times dilution of fungicide thiophanate-methyl for 8 h to kill the fungus 2 concentrations were randomly assigned into three replicates as main plot treatments, two endophyte statuses  were randomly assigned into the main plots as split plot treatments, and randomly selected plants were sampled at three times in two strips (two plants each time) as the split-split plots in two strips (3)2 concentrations ×3 sampling times), with three repetitions for each treatment and 12 plants for each repetition . There was a distance of 22 cm between strips  and 18 cm between plants within a strip. For split plot, plot size was 20 m2 with two surrounding-protection strips and two sampling strips. The strips of plants were 0.5 m apart from each other. Plants were grown for 5 months under natural conditions. After this period, plants were irrigated from 29 October to 28 November once a week for 4 weeks with 15 mL Ca(NO3)2 solution at three concentrations, 0 (CK), 25 and 50 mM, respectively. The herbage and roots of 6 different plants under each Ca(NO3)2 solution condition for either E+ or E- plants were randomly harvested from three repetitions in the morning on day 0 (before plant treatment), 14 and 28, respectively. Daily air temperature ranged from 3 °C to 14 °C during the day and −7 ° C to 2 °C during the night during the experiment according to the data from http://www.tianqihoubao.com/lishi/yuzhong.html  method . This method provides an estimate of metabolic activity in roots by reflecting aerobic respiration rates . To conduct this assay, root tips  were dipped in a solution containing five mL of 0.4% TTC and five mL of 1/15 M phosphate buffer solution (pH 7.0), and incubated at 37 °C for 1 h in the dark. Then, 2 mL of 1 M H2SO4 was added to the mixture to stop the reaction. The root was removed carefully, wiped and then ground with a small amount of ethyl acetate and quartz sand in a mortar to extract triphenyl formazan (TTF). The residue was washed 2–3 times with a small amount of ethyl acetate and added to the first extraction and made up to a volume of 10 mL. Root metabolic activity was measured using a spectrophotometer (SP-723) with an absorbance of 485 nm and using H2SO4 as a blank.Root metabolic activity was measured using the modified a) = (12. 72  × A663) (2.59 × A645), chlorophyll b (Cb) = (22.88 × A645) (4.86 × A663), total chlorophyll (Ca + Cb) = (8.02 × A663)+ (20.20 × A645), carotenoid = [ (3.27 × Ca) (104 × Cb)]/229.Chlorophyll and carotenoid contents were measured according to the modified method of Leaves were cut from the plant, weighed immediately , floated in water for 24 h to achieve turgidity , then oven-dried at 70 °C and weighed again . RWC of leaves was calculated according to the formula: [(FW DW)/(SW DW)] ×100%.Soluble sugars, sucrose, fructose and glucose were determined using the methods of 2SO4 and heated at 90 °C for 10 min. The mixture was immediately placed in tap water to cool and the absorbance measured at 620 nm. The concentrations of total soluble sugar were determined using glucose as the standard.For determination of soluble sugars, one mL of the filtered solution was mixed with 0.5 mL anthrone solution (1 g anthrone dissolved in 50 mL ethyl acetate) and five mL HFor determination of sucrose, 0.1 mL of the filtered solution was mixed with 50 µL of 2 M NaOH and incubated at 100 °C for 5 min, after which it was immediately cooled. The mixture was added to 0.7 mL of 30% HCl and 0.2 mL of 0.1% resorcinol, heated in a water bath at 80 °C for 10 min and after cooling absorbance was determined at 480 nm. The sucrose concentration was calculated using sucrose as the standard.2O, heated in a water bath at 80 °C for 10 min and cooled. Absorbance at 480 nm was determined. The fructose concentration was measured using fructose as the standard.For determination of fructose, 0.1 mL of the filtered solution was mixed well with 0.2 mL of 0.1% resorcinol and 0.7 mL H2SO4, and the absorbance was determined at 460 nm. The glucose concentration was determined using glucose as the standard.For analysis of glucose concentration, four mL of enzyme solution including 1 mg o-dianisidine dihydrochloride, 0.1 mg horseradish peroxidase  and 1 µL glycose oxidase  was placed at 30 °C until equilibrium after which two mL of the extracted sugar solution was added and mixed well for 5 min at 30 °C. The reaction was stopped by addition of eight mL of 10 M HStatistical analyses were performed with DPS software, Version 9.50. Data were presented as the mean ±  SE and means were compared by Fishers Least Significant Differences (LSD) test at 0.05 probability level. Homoscedasticity was evaluated by Levene’s test after some data were logarithmic, sine or cosine transformed. Correlation analysis was conducted between indicators measured and temperature using Spearman’s rho method (SPSS 16).P <  0.001) effects of Epichloë endophyte, calcium nitrate treatment, or treatment time were found on root metabolic activity of F. sinensis. In addition, there were some interactions between Epichloë endophyte, calcium nitrate treatment, and treatment time for root metabolic activity . On the 14th day, the presence of endophyte increased root metabolic activity comparing the E+ vs. E- plants, and E+ plants with 50 mM Ca(NO3)2 treatment [50Ca(NO3)2] had the highest level of root metabolic activity among all treatments. On the 28th day root metabolic activities of E- with 25 mM Ca(NO3)2 treatment [25Ca(NO3)2] were significantly higher than others (P <  0.05), and significant differences in E+ or E- plants were also observed (P <  0.05). When analyzing time shifts in root metabolic activities, samples except for E+ plants with 50Ca(NO3)2 had significantly higher root metabolic activity on the 14th day than on the 28th day, and lower on the 14th day than before treatment (P <  0.05).Highly significant (P <  0.001) effects of calcium nitrate treatment and treatment time were detected on total chlorophyll of F. sinensis. In addition, there were some interactions between Epichloë endophyte or calcium nitrate treatment, and treatment time for total chlorophyll  effects of Epichloë status on chlorophyll contents before treatment with calcium nitrate (3)2 were significantly (P <  0.05) higher than either E- plants or E+ plants with 50Ca(NO3)2. Furthermore, calcium nitrate addition significantly enhanced the chlorophyll contents in E- plants. On the 28th day the chlorophyll contents of E- plants with 25Ca(NO3)2 were the highest among all plants, and the chlorophyll contents of E+ plants supplied with 50Ca(NO3)2 were significantly higher than the other E+ plants (P <  0.05). In addition, there was time effect on chlorophyll contents (P <  0.05). The chlorophyll contents for the control and 50Ca(NO3)2-treated E- plants, and 50Ca(NO3)2-treated E+ plants were much lower on the 28th day than before and after 14 days treatment (P <0.05), however the chlorophyll contents in 25Ca(NO3)2-treated E- plants were greater on the 14th day than on the 28th day and before treatment (P <  0.05).Highly significant ( nitrate . Similarcontents . ChloropP <  0.001) effect detected on chlorophyll a/b ratio for F. sinensis (P <  0.05).Treatment time was the only highly significant 2 were significantly greater than those of untreated E+ plants (P <  0.05). Similarly on the 28th day, carotenoid contents of the control E+ plants were much greater than those of treated E+ plants and control E- plants, moreover calcium nitrate addition significantly increased carotenoid contents of the E- plants (P <  0.05). A significant time effect on carotenoid content was also observed (P <0.05). Carotenoid contents of treated E- plants before treatment were much lower than on the 14th day and 28th day (P <  0.05), control E- and E+ with 50Ca(NO3)2 for carotenoid contents were significantly (P <  0.05) decreased on the 28th day compared to on the day zero and the 14th day, and the carotenoid contents in control E+ sharply increased on the 28th day.Highly significant effect of treatment time was detected for carotenoids for P <  0.001) effect of treatment time was detected for relative water content of F. sinensis. In addition, there was significant Epichloë endophyte-by-treatment time interactions for RWC  as time passed after treatment (P <  0.05). On the 14th day RWC of E+ plants with 50Ca(NO3)2 were significantly (P <  0.05) less than those of the control E+ plants. In addition, on the 28th day RWC of E+ plants with 0 and 25Ca(NO3)2 were maintained at a significantly (P <  0.05) higher level than those of other plants.A highly significant  effects of treatment time or Epichloë endophyte were detected for soluble sugars in the shoots or roots of F. sinensis. In addition, there were some interactions between Epichloë endophyte, calcium nitrate treatment, and treatment time for soluble sugars in the shoots 2 significantly accumulated soluble sugars in the shoots (P <  0.05) compared to E- plants. On the 28th day the E+ plants had significantly higher soluble sugar concentrations in the shoots than the E- plants under control and 25Ca(NO3)2 (P <  0.05). However, calcium nitrate application greatly elevated soluble sugars in the roots in E- plants. In addition, there were substantial time changes in soluble sugar concentrations (3)2 treated E+ plants were much higher on the 28th day than before and after 14 days treatment (P <  0.05). Soluble sugar concentrations for 25Ca(NO3)2 treated E+ plants peaked on the 14th day (P <  0.05).Highly significant (trations  and 7. TP <  0.001) effects of treatment time were detected for sucrose levels of shoots and roots of F. sinensis. In addition, there were some significant interactions between Epichloë endophyte, calcium nitrate treatment, or treatment time for sucrose levels of shoots or roots 2 treatment when compared with control E- shoots (P <  0.05). On the 28th day sucrose concentrations of E+ shoots with 25Ca(NO3)2 were significantly greater than those of the control or 25Ca(NO3)2 treated E- plants (P <  0.05). In addition, on the 28th day control E+ roots accumulated higher sucrose concentrations compared to those of other plants, and E+ with 25Ca(NO3)2 increased more rapidly compared to those of E+ with 50Ca(NO3)2 (P <  0.05). With respect to the time series, the sucrose concentrations of roots were higher on the 28th day than on the 14th day and before treatment (P <  0.05).The highly significant (P <  0.001) effects of treatment time were detected for fructose concentrations of shoots and roots of F. sinensis. In addition, there were some interactions between Epichloë endophyte and treatment time for fructose concentrations of roots . The fructose concentrations of roots was significantly less on the 14th day than before treatment or on the 28th day (P <  0.05).The highly significant (P <  0.001) effects of treatment time were detected on the glucose concentrations of shoots and roots of F. sinensis (3)2 2 and control E+ were higher on the 28th day than on the 14th day and before treatment (P <  0.05), while other roots in glucose concentrations on the 14th day were much more than before treatment, and lower than on the 28th day (P <  0.05).The highly significant (sinensis . On the P <  0.05). The correlativity between temperature and root activity, and relative water content of leaf were significantly higher at the 0.01 level, the correlativity coefficients were 0.734 and 0.761, respectively. However soluble sugar concentration of shoot and sucrose concentration of root negatively correlated with temperature (P <  0.05), and soluble sugar concentration of root, sucrose concentration of shoot and glucose concentration of root negatively correlated with temperature (P <  0.01) with the Spearman correlations being 0.944, 0.682, and 0.866, respectively.Under field conditions, temperature and carotenoid concentration, shoot and root fructose concentrations, or glucose concentration of shoot were poorly correlated . There w4NO3). In this study we compared the changes in physiological performance and soluble sugar concentrations in 5-month-old F. sinensis E+ and E- plants when supplied with exogenous Ca(NO3)2 to elucidate the mechanism of plant survival during cold winter conditions.Low temperature induces phenotypic, physiological and biochemical changes in plants. Low temperature or growth inactivity of the plant may reduce uptake of nitrogen . Furthermore, endophytic fungi or exogenous calcium nitrate may be in effective in promoting root metabolic activity occurred early after cold treatment in field conditions (3)2 had higher root metabolic activity than E+25 on 28 d. It is thus likely that endophytes that inhabit aerial grass tissues of F. sinensis ineffectively enhanced the root metabolic activity under different Ca(NO3)2 treatments and low temperature. Ca concentration of roots in the present study need to be studied further.Low temperature is known to reduce root vigor . Consistd stress , Table 8nditions . In addiin vitro or arbuscular mycorrhiza or PRPP  are capable of alleviating chlorophyll degradation in plants at low temperature . However it has also been suggested that low carotenoid content was beneficial to pepper species exposed to low temperature and low irradiance . One study has shown that inoculation with Pseudomonas strains significantly enhances root/shoot biomass, relative water content, chlorophyll, anthocyanin, proline, proteins, and amino acids, which are adaptations to cold stress in wheat (3)2 in reducing RWC was greater for E+ plant than for E- . Epichloë endophyte may be playing a role in nutrient uptake and osmotic adjustment.In the experiments described in this paper, RWC was significantly reduced with decreasing temperature . Howeverin wheat . These fStupnikova et al., 2002). We found significant correlation between soluble carbohydrates and temperature 2 and fructose of roots under the control conditions on the 14th day, and on the 28th day significantly increased soluble sugar of shoots with control and 25Ca(NO3)2, glucose of shoots and sucrose of roots under the control conditions  induces more carbohydrate accumulations such as starch, glucose, fructose, sucrose, mannose, raffinose and maltose, in grapevine inoculated with oculated . Our datnditions  and 7. U3)2 application as in our study. Our results also indicated that calcium nitrate addition contributed to the accumulation of soluble sugars, sucrose, fructose or glucose in shoots and roots of both E+ and E- plants during long term cold stress 2significantly affected root metabolic activity, total chlorophyll, chlorophyll a/b ratio, and carotenoid. Epichloë endophyte and Ca(NO3)2 had no significant effect on chlorophyll a/b ratio. Some soluble sugar concentrations were affected by endophyte infection or interaction of the Epichloë endophyte and Ca(NO3)2 but not by Ca(NO3)2 alone. Further studies regarding soluble carbohydrate changes in addition to analysis of related enzyme activities, nutrient absorption and gene expression are necessary to elucidate the detailed mechanism of increased cold stress tolerance.In this work 10.7717/peerj.10568/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10568/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10568/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10568/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.10568/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.10568/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.10568/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.10568/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.10568/supp-9Supplemental Information 93)2 concentrations  were randomly allocated in three replicates as main plot treatments and two endophyte types  were randomly allocated in the main plots as split plot treatments. In each of the three sampling times , two plants were randomly sampled from a split-split plot 2 concentration).A diagram of treatment arrangement for a split-split-plot design, where three Ca(NOClick here for additional data file.


According to powder X-ray diffraction, electron diffraction and atomic resolution scanning transmission electron microscopy, Na9V14O35 adopts a monoclinic structure consisting of layers of corner- and edge-sharing VO5 tetragonal pyramids and VO4 tetrahedra with Na cations positioned between the layers, and can be considered as sodium vanadium oxovanadate Na9V104.1+O19(V5+O4)4. Behavior of Na9V14O35 as a positive and negative electrode in Na half-cells was investigated by galvanostatic cycling against metallic Na, synchrotron powder X-ray diffraction and electron energy loss spectroscopy. Being charged to 4.6 V vs. Na+/Na, almost 3 Na can be extracted per Na9V14O35 formula, resulting in electrochemical capacity of ~60 mAh g−1. Upon discharge below 1 V, Na9V14O35 uptakes sodium up to Na:V = 1:1 ratio that is accompanied by drastic elongation of the separation between the layers of the VO4 tetrahedra and VO5 tetragonal pyramids and volume increase of about 31%. Below 0.25 V, the ordered layered Na9V14O35 structure transforms into a rock-salt type disordered structure and ultimately into amorphous products of a conversion reaction at 0.1 V. The discharge capacity of 490 mAh g−1 delivered at first cycle due to the conversion reaction fades with the number of charge-discharge cycles.Na State-of-the art cathode materials for SIBs belong to two large groups: layered and 3-dimensional 3d and 4d metal oxides and polyanion structures   SAED pattern  and high discharge capacity of 480 mAh g−1 with three well-pronounced plateaus in the first discharge curve. The charge capacity of 55 mAh g−1 corresponds to extraction of three sodium atoms per unit cell, what perfectly matches the results of quantitative EDX analysis, showing the Na:V = 6:14 atomic ratio for the material charged to 4.6 V intercalation is provided by dQ/dV curves at the C/20 rate  Å, b = 5.0325(8)(1) Å, c = 20.781(2) Å, β = 109.166(5)°, V = 1502.1(4) Å3  Å3 . The dif 1500 Å3 . The absmaterial  are obvi9V14O35. Although the quality of the SXRD pattern is insufficient for the Rietveld refinement, the reflections still can be indexed with the P2/c unit cell of Na9V14O35, but with a much larger b-parameter resulting in an about 31% increase of the unit cell volume: a = 15.347(3) Å, b = 6.301(1) Å, c = 21.367(3) Å, β = 107.253(9)°, V = 1973.3(8) Å3 , but the expansion along the b-axis is huge and amounts to ~25.2%. This difference reflects the rigidity of the (010) layers formed by tightly interlinked VO5 square pyramids and VO4 tetrahedra, in which the expansion in the a-c plane occurs through elongation of the V-O bonds upon reduction of vanadium cations with increase in their ionic radius (r(V2+) = 0.79 Å, r(V3+) = 0.64 Å, r(V4+) = 0.58 Å, r(V5+) = 0.54 Å, CN = 6) [−1. This must cause structure instability, and indeed, the crystals with another symmetry were found in the 0.25 V-discharged material, as one can see from the SAED patterns and high-resolution HAADF-STEM images, which are typical for a disordered rock-salt (DRS) structure with F-centered cubic lattice, a unit cell parameter a ~ 4.7 Å  Å3 . The exp CN = 6) . Large i ~ 4.7 Å  and a Na ~ 4.7 Å . This ob pattern , but ano pattern , also ap density d.3,2 edge 4. Charging to 4.6 V vs. Na+/Na slightly shifts the V-L3,2 edge towards a higher energy loss resulting in EV = 515.4 eV and VV = +4.5 that corresponds to extraction of 3 Na per Na9V14O35 formula unit as deduced from the electrochemical data and EDX analysis . Upon discharge to 1 V and 0.25 V, the V-L3 edge onset energy is reduced to EV = 514.8 and 514.3 eV, respectively, providing VV = +3.9 and +3.5. It should be noted that the vanadium reduction from +4.5 to +3.5 upon discharge from 4.6 V to 0.25 V accounts for only ~250 mAh g−1 capacity that corresponds to the end of the first discharge plateau. Thus, the capacity of the second and third discharge plateaus should be attributed to a conversion reaction with the formation of the Na-rich phase. Unfortunately, the two-phase nature of the sample discharged to 0.1 V and strong Na inhomogeneity even within the same crystallite  and V2O5  at 550 °C for 14 h. Single-phase polycrystalline Na9V14O35 was prepared from 3.06 mmol NaVO3, 0.05 mmol V2O5 and 0.8 mmol V2O3 . The mixture of the initial reagents with the total weight of 0.5 g was ground thoroughly in an agate mortar, pressed into a 10 mm pellet, placed into an alumina crucible and sealed into a 16 mm quartz tube under dynamic vacuum of ~5∙10−3 mbar. The tube was heated with 120 °C/h heating rate to 650 °C, annealed for 50 h and cooled down with the furnace. The pellet was crushed, reground, pressed into a pellet and annealed at the same conditions for the second time.Nat at al. ,23. Init9V14O35 crystal structure was collected with a STOE Stadi P diffractometer   monochromator, MYTHEN detector, Mo-Kα1 radiation, λ = 0.7093 Å). A Huber G670 Guinier camera equipped with a Ge (111) monochromator  and an image plate detector and a Bruker D8 ADVANCE  equipped with energy-dispersive LYNXEYE XE detector were also used for control of the phase purity. Electrode composites scratched from the Al current collector were studied at DESY synchrotron source (SXRD) at the P02.1 beamline  with λ = 0.20736(1) Å. Diffraction data recorded by the Perkin Elmer 2D detector  were integrated and a wavelength was determined using reflection positions of LaB6 standard material. Rietveld refinement was performed using Jana2006 software [Powder X-ray diffraction (PXRD) pattern for the Rietveld analysis of the pristine Nasoftware .9V14O35, 10 wt.% carbon black (Super P) as a conductive additive and 10 wt.% polyvinylidenefluoride (PVDF) as a binder. Prior to preparation of a slurry, active material mixed with carbon black and moistened with acetone was subjected to 1 h treatment in a high-energy SPEX-8000 ball mill  to reduce particle size and perform an effective mixing of all components. Then, PVDF was dissolved in N-methyl-2-pyrrolidone and mixed with the active material into slurry. The resulting slurry was coated on an aluminum foil with the Dr. Blade applicator and then dried in vacuum at 85 °C. Next, 16 mm circular electrodes made from the coated foil were dried in vacuum at 110 °C overnight.Electrode composite was prepared from 80 wt.% Na4 in sulfolane:propylene carbonate mixed in 1:1 volume ratio was used as the electrolyte. Galvanostatic cycling with potential limitation was performed on a BioLogic potentiostat  at C/20, C/10 and C/5 rates, where 1C corresponds to the current density of 163 mA g−1 .The electrochemical cells were assembled in an argon-filled MBraun glove box ) with the residual oxygen and water content less than 0.1 ppm. A typical cell for galvanostatic cycling consisted of an electrode with the active material, a borosilicate glass fiber separator and sodium metal used as the counter electrode. Then, 1 M solution of NaClO9V14O35 powder in an agate mortar in acetone followed by depositing the suspension onto holey TEM grid with a Lacey/Carbon-supporting layer. The electrodes at different state of charge were removed from electrochemical cells, double-washed in dimethyl carbonate (DMC) and scratched from the Al foil. All manipulations were performed in Ar-filled glove box followed by transportation to the TEM column by means of a dedicated vacuum holder, completely avoiding contact with air and moisture. Selected area electron diffraction (SAED) patterns, high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and energy dispersive X-ray (EDX) compositional maps were taken with an aberration-corrected FEI Titan G3 transmission electron microscope  operated at 200 kV. The electron energy loss spectra (EELS) were registered in a STEM mode on a Titan Themis Z TEM  equipped with a Gatan Quantum ERS/966 P spectrometer  and operated at 200 kV. Energy dispersion of 0.025 eV per channel was used; the energy resolution measured by full width at half maximum of zero-loss peak was 0.125 eV.The morphology was characterized with a Quattro S ESEM scanning electron microscope . Samples for transmission electron microscopy were prepared by crushing the Na9V14O35 (η-NaxV2O5) has been synthesized by a solid-state route in an evacuated sealed silica tube and tested as electroactive material for Na half-cells. Being charged to 4.6 V vs. Na+/Na, almost 3 Na can be extracted per Na9V14O35 formula unit, resulting in a charge capacity of about 60 mAh g−1. Upon discharge below 1 V, Na9V14O35 uptakes Na up to the Na:V = 1:1 atomic ratio that is accompanied by a drastic increase of the separation between the layers of the VO4 tetrahedra and VO5 tetragonal pyramids, and a volume increase of about 31%. The induced structure instability triggers a transformation of the ordered layered Na9V14O35 structure into a rock-salt type disordered structure. Ultimately, the amorphous products of a conversion reaction are formed at 0.1 V, delivering the discharge capacity up to 490 mAh g−1, which, however, quickly fades with the number of charge-discharge cycles.Na


The effects of MXene on non-isothermal crystallization and polymorphic behavior of β-iPP/MXene composites were comparatively studied. The non-isothermal crystallization kinetics indicates that for all samples, the lower cooling rates promote composites to crystallize at higher temperatures. When MXene and β-Nucleating agent (β-NA) are added separately, the crystallization temperature of composites shifts towards higher temperatures at all cooling rates. When MXene and β-NA are added simultaneously, the composite shows different cooling rate dependence, and the effects of improving crystallization temperatures is more obvious under rapid cooling. The activation energy of four samples iPP, iPP/MXene, iPP/β-NA, and iPP/MXene/β-NA were −167.5, −185.5, −233.8, and −218.1 kJ/mol respectively, which agree with the variation tendency of crystallization temperatures. The polymorphic behavior analysis obtained from Differential Scanning calorimetry (DSC) is affected by two factors: the ability to form β-crystals and the thermal stability of β-crystals. Because β-crystals tend to recrystallize to α-crystals below a critical temperature, to eliminate the effect of β-α recrystallization, the melting curves at end temperatures Tend = 50 °C and Tend = 100 °C are comparatively studied. The results show that more thermally unstable β-crystals would participate in β-α recrystallization with higher cooling rates. Moreover, thermal stability of β-crystals is improved by adding MXene. To further verify these findings, samples of three different thermal conditions were synthesized and analyzed by DSC, X-Ray Diffraction (XRD), and Polarized Light Optical Microscopy (PLOM), and the results were consistent with the above findings. New understandings of synthesizing β-iPP/MXene composites with adjustable morphologies and polymorphic behavior were proposed.In this study, one of the commonly used MXene (Ti Isotactic polypropylene (iPP), firstly synthesized by Natta et al. in the laboratory and industrialized in 1957 ,3. iPP in+1XnTx, where M is an early transitional metal element, X is carbon (C) or nitrogen (N) element, Tx represents the various surface terminations such as fluorine, hydroxyl, and/or oxygen atoms [n+1AXn where n generally varies between 1 and 3, and A is an A-group element (mostly groups 13 and 14) such as Al, Si, P, S, Ga, etc. [3C2Tx is one of the most promising materials and has been widely studied and researched [3AlC2 can lead to superior behavior in electronic conductivity up to 20,000 S/cm, expanding the applications of MXene. Several studies have shown that Ti3C2Tx acts as a potential candidate for fillers in polymer composites. Ling et al. firstly synthesized polyvinyl alcohol (PVA)/ Ti3C2Tx composites with high electrical conductivities and achieved a 300% increase in tensile strength compared with pure PVA films when introducing 40 wt% Ti3C2Tx. Yi et al. [3C2Tx composites and investigated the mechanical properties and crystallization behavior. With the addition of 0.5 wt% Ti3C2Tx, the elongation at break was improved 5.9-fold (up to 131.6%), and the crystallinity was also improved due to the heterogeneous nucleation effect. Zhang et al. [3C2Tx composite and achieved maximum tensile strength at 0.75 wt% concentration of Ti3C2Tx. Wan et al. [3C2Tx composite film with balanced shielding performance and mechanical property by introducing poly/poly(styrenesulfonate) (PEDOT/PSS) treated in sulfuric acid. The synthesized Ti3C2Tx composite film exhibited EMI SE around 40.5 dB and tensile strength around 38.5 MPa with thickness of 6.6 μm.In the past decade, two-dimensional (2D) materials such as graphene and black phosphorus have drawn significant attention because of their unique structure. They possess superior properties showing potential application in fields such as energy storage, supercapacitor, composites, etc. Recently, Ahmadivand et al.  have repen atoms . MXenes Ga, etc. ,20,21. MGa, etc. ,22. Amonsearched ,23. Likesearched , high elsearched ,26, largsearched , hydrophsearched , excellesearched , and havd up to 1.6%, and n et al.  prepared3C2Tx composites at different concentrations. They found that the crystallization process is promoted below 0.5 wt% Ti3C2Tx due to the heterogeneous nucleation effect, but slows down with further increasing Ti3C2Tx content because of the rigid confinement network [3C2Tx composites. When 0.5 wt% Ti3C2Tx was added, the peak crystallization temperature and crystallization rate both increased. Once the concentration of Ti3C2Tx reached 1 wt%, the crystallization process was retarded, which might be caused by the confined network [While these works focus on improving the physical properties of polymer/MXene composites, the crystallization behavior of composites is still less reported. Huang et al. investigated the crystallization behavior of Poly(ethylene oxide) (PEO)/Ti network . Similar network .3C2Tx and fabricates β-iPP/MXene composites to investigate the non-isothermal crystallization kinetics and polymorphic behavior of composites by differential scanning calorimetry (DSC), X-Ray Diffraction (XRD), and polarized light optical microscopy (PLOM). New understandings in preparing the β-iPP/MXene composites with tunable morphologies and polymorphic behavior were also proposed.It is well-known that crystallization behaviors can affect the physical and mechanical properties of composites. Moreover, the practical manufacturing process is usually proceeded under non-isothermal crystallization conditions, understanding the non-isothermal crystallization is of great importance. To our best knowledge, the roles of MXene in the polymorphic behavior, crystalline morphologies of β-iPP at varied cooling conditions had not been investigated yet. Thereby, this work chooses the common MXene Ti3AlC2  was purchased from 11 Technology Co. Ltd. . Lithium fluoride powders  were purchased from Aladdin Bio-Chem Technology Co. Ltd. . Concentrated hydrochloric acid (HCl) with 37 wt% concentration was obtained from Chengdu Kelong Chemical Reagent Factory .The precursor MAX phase Ti−1 and the average isotacticity was around 97.6%. The β-nucleating agent (β-NA) used in the work was WBG-II, which is a commercial nucleating gent. The general formula of WBG-II is CaxLa1−x(LIG1)m(LIG2)n, where LIG1 and LIG2 are dicarboxylic acid and amide-type ligands [The isotactic polypropylene resin (tradename T38F) was obtained from Lanzhou PetroChemical Corp. . The molecular weight was 347,000 gmol ligands ,36.3AlC2, HCl and LiF were used to in situ form HF. The schematic diagram of etching Ti3AlC2 into Ti3C2Tx is indicated in 3AlC2 was immersed slowly into the solution and kept under magnetic stirring for 24 h at a temperature of 40 °C to allow full reaction. When the etching process was completed, the mixed solution was washed with distilled water and centrifuged at 8000 rpm for 10 min to separate the supernatant from the Ti3C2Tx sediment. This washing–centrifugation cycle was repeated until the pH value of the supernatant reached around 6. The sediment was immersed in distilled water for ultrasonication in the ice bath water for 2 h. The mixture was then centrifuged again to collect the final supernatant and dried under vacuum.To selectively etch the precursor Ti3C2Tx and β-NA WBG-II were firstly mixed with iPP resin by the Mini-Lab extruder at a temperature of 200 °C and screw speed of 80 rpm to prepare two masterbatches with 2.5 wt% concentration. In the second step, the masterbatches were mixed again with iPP resin to prepare four different samples. The prepared samples were then pressed by a pressure molding machine under 190 °C and 8 MPa for further characterization. To benefit the following discussion, the prepared samples were named as neat iPP, iPP/MXene, iPP/β-NA, and iPP/MXene/β-NA. The concentrations of Ti3C2Tx and WBG-II were fixed at 0.5 wt% and 0.1 wt%, which were chosen based on some previous studies [The composites were prepared by melt blending via a Mini-Lab Extruder . The Ti studies ,37,38. TαCu K radiation (λ = 0.154 nm) with a voltage of 40 kV and filament current of 40 mA. To measure the spectra of Ti3AlC2 and Ti3C2Tx powders, the scanning range was set to 2θ = 2 − 80° and the scanning rate was 10°/min. Before measuring iPP composites, the samples were firstly hot molded into sheets with 1 mm thickness, and the scanning range was 2θ = 5 − 40° at a scanning rate of 2°/min. The relative content of the β phase (kβ) could be calculated from the XRD spectra via the following equation [βH(300) denotes the intensity of (300) reflection of β phase. αH(110), αH(040), and αH(130) denote intensities of the three strongest reflections of α phase.XRD measurements were performed using a diffractor  with a equation ,39:(1)kβ3AlC2 and Ti3C2Tx powders were observed by SEM  equipped with EDS. The voltage was 5 kV and working distance was 4.9 mm.The morphology of Ti3C2Tx, the sample was dispersed in distilled water under ultrasonication for 10 min. Then, the solution was dropped on a copper grid for observation.TEM characterization was conducted on a Tecnai G2 F20 S-TWIN  with an accelerating voltage of 200 kV. To observe the structure and dispersion of Ti−1. For each experiment, the standard procedure was applied as follows: 3–5 mg sample was weighted and heated to 200 °C to erase the previous thermal history. Then, the sample was cooled to end temperature 50 °C at a cooling rate of 5, 10, 20, 30, and 40 °C/min, respectively, and reheated to 200 °C at 10 °C/min to analyze its crystallization and following melting behavior. To ensure the accuracy of the data, the sample was repeatedly tested five to eight times to obtain the average value.A Mettler Toledo DSC3  differential scanning calorimetry was used to perform the nonisothermal crystallization experiments under a continuous nitrogen flow of 50 mL mintX) as a function of temperature (T) can be calculated by the following equation [T0 and T∞ are the onset and end crystallization temperature, and dH/dT is the heat flow rate. In nonisothermal crystallization, the time scale t can be transformed from the temperature T by the equation:Xt versus time t can be plotted.The relative degree of crystallinity XtXt is the relative degree of crystallinity, t is crystallization time, n is the Avrami exponent, and k is a crystallization rate constant. n usually varies between 1 and 4 and can be influenced by the combined effect of nucleation and growth [Avrami equation is generally applied to analyze the isothermal crystallization kinetics by the equation:d growth . The doun and lnk can be calculated by fitting the experimental data. Since the Avrami equation describes the isothermal crystallization process, Jeziorny suggested a method to modify the parameter k by introducing the cooling rate By plotting the graph of β phase (βc) was calculated by the following equation:H/ΔuH. ΔH and ΔuH are the apparent and complete crystalline heats of fusion respectively. The value of ΔuH for 100% crystalline iPP is 209 J/g [The relative percentage crystallinity of  209 J/g ,44,45.The crystalline morphologies of samples were investigated by PLOM  coupled with a hot-stage . A small piece of the sample was cut and placed between glass covers, melted at 200 °C  for 5 miθ ≈ 9.6° to 6.4° after etching, suggesting an expanded interlayer distance. The disappearance of the most intense (104) diffraction peak at 2θ ≈ 39°, which is representative of Ti3AlC2, further confirms the complete etching.As shown in 3AlC2 and Ti3C2Tx. As shown in 3AlC2 exhibits a compact layered structure in which the flakes were closely stacked, and this particular structure can often be observed in ternary carbides [2 during the etching process due to the exothermic reaction between HF and Al [SEM was conducted to observe the morphologies of bulk Ticarbides . After tF and Al ,49.c), onset and end crystallization temperatures , and crystallization peak width (Tconset—Tcend) are plotted in conset—Tcend, the greater the crystallization temperature range [The cooling curves of the four samples are plotted in re range ,50.c, Tconset, and Tcend. In other words, a lower cooling rate enables the sample to crystallize at a higher temperature. Moreover, the crystallization peak width Tconset—Tcend decreases with a lower cooling rate, which indicates that the polymer chains get sufficient time to rearrange into crystalline phase. The variation trend for the four samples is slightly different. From c, Tconset, and Tcend for neat iPP are the lowest, while the crystallization peak width is the largest. Neat iPP has the weakest crystallization ability among all samples, so it crystallizes at lower temperatures. When adding MXene, Tc, Tconset, and Tcend increase, but the crystallization peak width Tconset—Tcend remains nearly unchanged, suggesting that the crystallization ability is improved at all cooling rates by adding MXene. The improved crystallization ability of iPP/MXene might be attributed to the heterogeneous nucleation effect of MXene [c, Tconset, and Tcend of iPP/β-NA are the highest while Tconset—Tcend is the smallest, indicating that the addition of β-NA can greatly improve the crystallization ability of iPP, and the effect is more significant than the addition of MXene. This finding is consistent with the previous research results [Firstly, it is found that for all samples, the lower the cooling rate is, the larger the value of Tof MXene . When β- results .c and Tconset—Tcend are both similar to iPP/MXene, indicating that under the combined effect of MXene and β-NA, the crystallization ability is not further improved. However, when the cooling rate increases (>10 °C/min), Tc and Tcend are higher than iPP/MXene but lower than iPP/β-NA, and the crystallization peak width is lower than iPP/MXene. This finding suggests that adding MXene and β-NA improve the crystallization temperature while narrowing the crystallization peak, and this effect is found more obvious under rapid cooling.Interestingly, when MXene and β-NA are added simultaneously, the crystallization parameters of iPP/MXene/β-NA exhibit a different cooling rate dependence from other samples. At a lower cooling rate (≤10 °C/min), TtX) as a function of time curves at different cooling rates were plotted in 1/2 is calculated when tX is 50%. The value of t1/2 can be used as a measure of crystallization rate and ability. In general, the larger the value of t1/2 is, the longer the time needed for the crystallization process. The plotted curves in 1/2 of neat iPP is higher than the other three composites at all cooling rates, suggesting its crystallization rate is the lowest. t1/2 of iPP/MXene/β-NA at lower cooling rates is almost the same as that of iPP/β-NA, while increases significantly at higher cooling rates, proving that the crystallization ability of iPP/MXene/β-NA is not as good as iPP/β-NA. This result agrees with findings from DSC cooling curves.The cumulative relative degree of crystallinity  is calculated by Equation (7) and shown in Figure 11. Figure 11a shows that at Tend = 50 °C, βc gradually decreases with increasing cooling rate, which seems that a lower cooling rate is more beneficial for the formation of high content β-crystals. However, the βc calculated from DSC is affected by two factors. One is the ability to form β-crystals, i.e., the amount of β-crystals. The second factor is the thermal stability of β-crystals. According to the study of Varga et al. [end = 100–105 °C) would recrystallize into α-iPP in the subsequent melting behavior. The exothermic recrystallization peak of β-crystals coincides with the endothermic melting peak, which would interfere with the accuracy of βc on DSC curves. To comparatively study the polymorphic behavior at different cooling rates, we compared the melting curves at two end crystallization temperatures Tend = 50 °C and Tend = 100 °C, the melting curves and calculated βc are plotted in The relative percentage crystallinity of β-phase , β (300), α (040), and α (130) diffractions [β value calculated from XRD patterns at all cooling rates is higher than βc value calculated from DSC melting curves. Apart from that, when the cooling rate increases, the kβ decreases from 91.3 to 82.8% in iPP/β-NA and from 90.0 to 79.1 % in iPP/MXene/β-NA. This trend also agrees with findings from DSC melting curves.The XRD results of iPP/β-NA and iPP/MXene/β-NA were further compared in ractions ,59,60. Send = 50 °C and Tend = 100 °C show different variation tendency. When the crystallization end temperature Tend = 50 °C, a lower cooling rate leads to more formation β-crystals. But this trend is reversed at Tend = 100 °C because more unstable β-crystals would participate in β-α recrystallization with higher cooling rates. Moreover, the experimental phenomenon that kβ calculated from XRD is higher than βc calculated from DSC at all cooling rates further proves the β-α recrystallization. The study paves a method to prepare β-iPP/MXene composites with adjustable crystallization and polymorphic behavior, which can provide new insight to develop high performance iPP/MXene composites with excellent mechanical properties in future work.The effects of MXene on nonisothermal crystallization behavior β-iPP/MXene composites were investigated. The DSC analysis shows that the crystallization ability of the samples follows the order iPP/β-NA, iPP/MXene/β-NA, iPP/MXene, iPP. When MXene and β-NA are added separately, the crystallization temperature of composites increases with lower cooling rates. However, when MXene and β-NA are added simultaneously, they tend to compete with each other. Due to the low thermal stability of β-crystals, the melting behaviors at two end temperatures T


Root hypoxia has detrimental effects on physiological processes and growth in most plants. The effects of hypoxia can be partly alleviated by ethylene. However, the tolerance mechanisms contributing to the ethylene-mediated hypoxia tolerance in plants remain poorly understood.Populus tremuloides) seedlings. Ethylene enhanced net photosynthetic rates, transpiration rates, and root hydraulic conductance in hypoxic plants. Of the two subgroups of PIPs (PIP1 and PIP2), the protein abundance of PIP2s and the transcript abundance of PIP2;4 and PIP2;5 were higher in ethylene-treated trembling aspen roots compared with non-treated roots under hypoxia. The increases in the expression levels of these aquaporins could potentially facilitate root water transport. The enhanced root water transport by ethylene was likely responsible for the increase in leaf gas exchange of the hypoxic plants.In this study, we examined the effects of root hypoxia and exogenous ethylene treatments on leaf gas exchange, root hydraulic conductance, and the expression levels of several aquaporins of the plasma membrane intrinsic protein group (PIP) in trembling aspen (PIP2;4 and PIP2;5 in hypoxic roots of trembling aspen. The results suggest that ethylene facilitates the aquaporin-mediated water transport in plants exposed to root hypoxia.Exogenous ethylene enhanced root water transport and the expression levels of The online version contains supplementary material available at 10.1186/s12870-021-02995-7. Contrarrlogging , 4. Evenic rates . Althougtoxicity .2 [3 [2 [Root hypoxia commonly alters plant water relations and may cause wilting , 8. This2 , NH3 [142 [3 [2 . Plant a2 [3 [2 . In high2 sensing pathway was proposed in Arabidopsis, which may regulate the aquaporin activity [2 [2 [Aquaporins mediate root hydraulic adjustments in response to environmental factors . Aquaporactivity . Althougactivity , 21. On activity , 23, CO2ivity [2 , and O2 ty [2 [2  in plantArabidopsis was increased by ethylene through the enhanced C-Terminal phosphorylation of AtPIP2;1 [The inhibitory effects of hypoxia on root water transport in some plants could be partly reversed by the exposure of roots to exogenous ethylene . EthylenAtPIP2;1 . While tP. tremuloides) seedlings to shed more light on the regulation of aquaporin-mediated water transport by O2 deficiency and ethylene. It was hypothesized that the effects of ethylene on root hydraulics under hypoxic conditions involve changes inthe aquaporin gene expression.In the present study, we examined the effects of root hypoxia and exogenous ethylene application on gas exchange, water transport and the expression of ERF and aquaporin genes in trembling aspen (Pn), transpiration rate (Tr) and stomatal conductance (gs)  Table ; Fig. 1.Pn and gs. Hypoxia treatments significantly decreased the light saturation point (Im) and the level of Pn at light saturation (Pm)  Table a. Ethylets Table . When exne Table . Light rons Fig. b.Kr)  Table ; Fig. 2.ERFs)  Table . Signifies Table . EthylenPIPs expression level, hypoxia significantly decreased the relative transcript abundance of PIP1;2 and PIP2;2, but significantly increased the relative transcript abundance of PIP2;1 and PIP2;5  seeds were collected and identified by Dr. Wenqing Zhang  from the North Saskatchewan River valley . Authorization is not required for seed collection in this area. Seeds were sterilized twice with 1 % (v/v) sodium hypochlorite and germinated in horticultural soil in a controlled growth room with 18 h photoperiod, 22/18oC (day/night) temperature, 400 µmol m− 2 s− 1 photosynthetic photon flux density and 50 % relative humidity. When five-weeks old, the plants were removed from pots and after rinsing the roots free of soil, they were transferred to 40 L polyethylene containers (60 × 40 × 20 cm) filled with a quarter-strength aerated modified Hoagland’s solution [2 concentration of about 7.5 mg L− 1 throughout the experiment. The remaining 12 plants were subjected to hypoxia treatment by flushing N2 gas  for 30 min through the solution until reaching an O2 level of 2 mg L− 1. When the desired O2 concentration was reached, the plants where left in the stagnant solution for one week. The dissolved O2 concentration, which was monitored during the experiment with the YSI-5000 O2 electrode  and declined in the stagnant solution to less than 1 mg L− 1 over the one-week treatment period  were treated with exogenous ethylene at the beginning of the treatments. Ethylene was supplied to the hydroponic solution from the compressed gas cylinder through a tube stretched to the bottom of plastic bottles [2, and the vacutainers were shaken at 1,400 RPM for 5 min. From the headspace, 100 µl gas samples were collected and injected into the Varian 430-GC gas chromatograph  equipped with a HP Plot-Q column . The concentration of dissolved ethylene was calculated based on the Henry’s Law and a constant of 4.9 × 10− 3 M atm− 1 was used [− 1.Half of the plants in the aerated control and hypoxia treatments , stomatal conductance (gs) and transpiration rate (Tr) using a LI-6400XT Portable Photosynthesis System with a 2 × 3 cm2 red-blue light chamber . The reference CO2 concentration was set to 400 µmol mol− 1; the flow rate was 200 µmol s− 1. The leaf chamber temperature was maintained at 20 °C, and PPFD was 400 µmol m− 2 s− 1 of the red-blue light spectrum. To evaluate the effects of treatments on leaf gas exchange under varying light conditions, light responses of Pn and gs were established by setting an automated program with a starting photosynthetic photon flux density (PPFD) of 1500 µmol m− 2 s− 1, followed by 1200, 1000, 800, 500, 300, 200, 100, 50, 20 and 0 µmol m− 2 s− 1. Parameters were auto-logged when Pn reached steady state. Light saturated Pn (Pm) and light saturation point (Im) were estimated by fitting the data to a modified rectangular hyperbole model [One fully extended leaf was randomly selected from each of the six plants per treatment during 9:00 to 12:00 h to measure net photosynthesis rate (le model .Kr) was measured with a high-pressure flow meter . During the measurements of six plants in each treatment, roots were kept in the respective treatment solutions. Linear regression between the supplied pressure and flow rate was obtained by applying increasing pressure to the roots, and the slope was calculated as Kr [Root hydraulic conductance . First strand of cDNA was synthesized from 1 µg total RNA using a Reverse Transcription Kit (Qiagen). Quantitative RT-PCR was employed to analyze RNA expression using JIP1 (AJ407583.1) and ACTIN1 (Potri.001G309500) as reference genes, which did not change significantly across all tested samples (P = 0.36). The transcript abundance of PIP1;1 (AJ849323.1), PIP1;2 (AJ849322.1), PIP2;1 (AJ849324.1), PIP2;2 (AJ849325.1), PIP2;3 (AJ849326.1), PIP2;4 (AJ849327.1) and PIP2;5 (AJ849328.1) were determined. These PIPs have been proven to be functional and stress responsive in poplar plants [ERF17, ERF18, ERF35, ERF71 and ERF76, was determined since these genes were reported to be induced by ethylene in P. tremula × tremuloides [−∆Ct method. The primers for trembling aspen PIPs [ERFs [After one week of treatment, primary roots  of four to six plants from each treatment were collected and kept in liquid nitrogen before being transferred to -80r plants . The tramuloides . The relPs [ERFs  were desoC. The anti-PIP1 antibody (R-4445) was raised against an amino peptide from ZmPIP1;5 and the anti-PIP2 antibody (R-2493) was raised against an amino peptide from ZmPIP2;4 and was shown to recognize PIP1s and PIP2s in maize and Larix laricina (Provided by Dr. F. Chaumont) [Zea mays) PIPs. The membrane was rinsed 5 times for 5 min with TBST and then incubated in GAR IgG antibody (1:2000)  for 1 h at room temperature. After rinsing the membrane with TBST 5 times for 5 min, alkaline phosphatase (Bio-Rad) was conjugated to detect signals.SDS gel electrophoresis was performed to separate proteins (10 µg) extracted from roots after one week of treatment. Gel was transferred to a PVDF membrane  by electroblotting at 100 V for 1 h. The membrane was blocked in 1 % BSA in TBST buffer  for 3 h and then incubated with the anti-PIP1 (1:500) or -PIP2 (1:1000) primary antibody overnight at 4haumont) , 52. TheThe means and standard errors of aerated plants (A), aerated plants treated with ethylene (AE), hypoxic plants (H), and hypoxic plants treated with ethylene (HE) were calculated for each of the measured parameters. Two-way ANOVA was used to examine the effects of aeration (aerated vs. hypoxia treatments) and ethylene treatments (with or without ethylene). The data that did not meet the ANOVA assumptions of normality of distribution and homogeneity of variance were transformed with a square-root function. Comparisons between different treatment means were conducted using LSD test (α = 0.05).Additional file 1: Table. S1. Effects of hypoxia and ethylene on the parameters determined in trembling aspen (P. tremuloides). Table S2. List of qRT-PCR primers used in this study. Figure S1. Light response of net photosynthesis and stomatal conductance in trembling aspen (P. tremuloides). Figure S2. Root length and shoot length in trembling aspen (P. tremuloides). Figure S3. Root porosity in trembling aspen (P. tremuloides). Figure S4. Full length original Western-blot pictures of PIP1s (a) and PIP2s (b) in trembling aspen (P. tremuloides). Figure S5. Transcript abundance of endo-ethylene synthesis related genes  in trembling aspen (P. tremuloides). Figure S6. Dissolved oxygen concentration during one-week treatments in trembling aspen (P. tremuloides).


E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant  These modifications through subsequent signal transductions pathways lead to decreased TLR-4 expression and increased IL-10 production (via the conversion of bilirubin IXα (BRIXα) to BVIXα and back via BVR (Pseudomonas aeruginosa whose heme oxygenase (HemO) degrades heme with the release of BVIXβ and -δ  produces biliverdin IXα (BVIXα) a metabolite well known for its anti-inflammatory and immunosuppressant functions through indirectly downregulating pro-inflammatory cytokines (TLR-4)  and upreoduction ,b. In adoduction  as well  via BVR . HOs areβ and -δ . Recentltal bile  and the tal bile . While ttal bile , the orital bile .P. aeruginosa the iron regulated HemO oxidatively cleaves heme to release BVIXβ and BVIXδ  E. coli strain Nissle 1917 (EcN)  . The EcN17 (EcN) . This st17 (EcN) . The comA mutant  . Similar precautions for LC-MS/MS BVIX isomer validation using LC-MS grade (Fisher Optima and Thermo Scientific for DMSO) reagents and solvents. Specific medias, columns, and instruments are listed below where appropriate.Escherichia coli Nissle(T7) cells were obtained from the Frankenberg-Dinkel laboratory. EcN(T7) electrocompetent cell were made fresh as previously described (600 = 0.05 and grown with shaking (210 rpm) at 37°C until the OD600 reached 0.5–0.6 (mid-log phase). The culture was then centrifuged at 5,000 × g at 4°C for 5 min and pellets were washed with 25 mL chilled 10% glycerol and the wash repeated twice more. Following the washing steps the culture was recentrifuged and suspended in 250 μL of 10% glycerol to a final 100x lower volume from the initial culture. 30 μL aliquots of the final cell volume (250 μL) were used for electroporation or stored at −80°C for up to 3 months. Electroporation was performed as previously described  prepared in DMSO (10–100 μL) and shaken at 250 rpm overnight at 25°C. Cultures were pelleted  in 50 mL conical tubes, and the supernatants were collected for BVIX extraction or stored at −80°C for up to 1 month. Pellets were thawed, resuspended in Milli Q water and 2 μg/mL DNase I, sonicated at 100% amplification for 5 secs on and 15 secs off for 2 min, and run on a 12% SDS PAGE gel at 200V for 30 min as previously described with slight modifications . After sample application, the column was washed with 40 mL 0.1% TFA, 40 mL ACN:0.1% TFA (20:80), 20 mL methanol:0.1% TFA (50:50) and eluted with 15 mL methanol. Alternatively, for smaller batches the isomers were purified over a 3cc (Waters) C18 Sep-Pak column equilibrated as above scaling back the washes and elution volumes x10. The supernatant was centrifuged  and the remaining solution containing purified BVIX isomers was collected. Purified BVIX isomers were diluted with an equal volume of water and extracted into CHCl3 (3–5 ml) washed with ×3 with 5 ml H2O to remove any remaining acid. The CHCl3 layer was collected, and speed vacuumed dry and stored at −80 °C prior to HPLC separation.Biliverdin IX isomers were extracted as previously described  with sliTM 3000 HPLC) on an SynergiTM 4 μm Fusion-RP 80 Å C18 Column (250 × 10 mm) at a flow rate of 3 mL/min. The mobile phase consisted of solvent A, H2O:0.1% formic acid, and solvent B, ACN:0.1% formic acid. A linear gradient was run starting at 64% A and 36% B and ending with 0% A and 100% B over 20 or 30 mins. BVIX isomer retention times were 9.9, 10.8, and 11.6 min for BVIXα, BVIXδ, and BVIXβ isomers, respectively, for the 20 min linear gradient and 9.7, 12.1, and 13 min, respectively for the 30 min linear gradient. Separated isomers were then extracted into choloroform as described above, speed vacuumed dry and stored at −80°C. The final yield of each BVIX isomer was calculated from their respective millimolar extinction coefficients (εmM) at 376 nm and 665 nm in methanol as previously described  to remove any particulates. The BVIX isomers were then separated by HPLC  BVIX production method, TotalC for BVIXβ, and -δ isomers were calculated based on HemO BVIX ratio of 30:70, respectively. Molar extinction coefficients of BVIXα, -β, and -δ isomers in DMSO were obtained using 380 ± 1/658 ± 3 nm, 389 ± 1/651 ± 1 nm, and 386 ± 2/645 ± 5 nm, respectively, (n = 3) based on the UV spectrum , and centrifuged at 14,000 rpm for 5 min at 4°C to remove particulates. BVIX isomers (2 μL) were analyzed on a Waters TQ-XS triple quadrupole mass spectrometer with AQUITY H-Class UPLC fitted with an Ascentis RP-amide 2.7-mm C18 column (10 cm × 2.1 mm) at 30oC with a flow rate of 0.4 mL/min. The mobile phase consisted of solvent A, H2O:0.1% formic acid, and solvent B, ACN:0.1% formic acid. The initial gradient was 64% A and 36% B followed by 5 min at 55% A and 45% B, 8 min at 40% A and 60% B, 8.5 min at 5% A and 95% B, and 10 min 64% A and 36% B. Using multiple-reaction monitoring (MRM), the BVIX precursor ions were detected at 583.21 and the major ions following fragmentation for BVIXα, BVIXβ, and BVIXδ isomers were 209.2/296.6, 343.1, and 402.2 m/z, with collision energies of 38, 36, and 30 V, respectively. The source temperature was set to 150°C, the capillary voltage to 3.60 kV, and the cone voltage to 43 V. Purity of the BVIX isomers was determined by the ratio of the integration of the isomer peaks to the total integration of the LC chromatograms.Following HPLC separation the respective BVIX isomers were confirmed by LC-MS/MS as previously described with slight modification . BrieflyE. coli BL21(DE3) (Expression of HemO in EcN(T7) was performed across a range of induction temperatures, incubation times, and shaking conditions . The optL21(DE3) .The C18 BVIX isomer extraction was performed in semi-darkness to protect the isomers from photoisomerization. The C18 eluted BVIX fraction was centrifuged to remove any excess acidified proteins and the UV-visible spectrum of the fraction was recorded. The absorption spectrum while characteristic of BVIX with a broad absorbance at 680 nm  did appe2O:0.1% formic acid, and solvent B, ACN:0.1% formic acid can achieve baseline separation of the BVIXβ and -δ isomers,  has been used as a means of producing BVIXδ and BVIXβ (E. coli BL21 (DE3) strain was employed to produce BVIXδ and BVIXβ, but the yields obtained were lower than that of the chemical coupled oxidation of heme (<0.05%), even when supplementing cultures with exogenous heme or the heme precursor δ-aminolevulinic acid to promote heme biosynthesis. Given the ability of the engineered EcN(T7) to naturally transport heme we coupled this to the over expression of HemO in an attempt to maximize BVIX yields. Interestingly, following centrifugation of the cultures, we saw no green coloration in the pellet as was observed on HemO expression in E. coli BL21 (DE3) (P. aeruginosa (PAO1) also secretes BVIXα into the supernatant (E. coli BL21 (DE3) strain where the majority of BVIX remains in the cell pellet (E. coli BL21 (DE3) cells (E. coli BL21(DE3), it should be noted that the authors utilized a modified cyanobacterial ho1 gene and performed the expression in a large scale Bioreactor (3) extraction steps of which both are toxic and carcinogenic (Previous work utilizing HemO over-expression in nd BVIXβ . Similar21 (DE3) . However21 (DE3) , suggest21 (DE3) . We haveernatant , suggestl pellet . The saml pellet . Interes3) cells . Further3) cells . Therefooreactor . Furtherinogenic .2O 0.1% formic acid. Previous ACN-based HPLC methods for the separation of the BVIX isomers have reported baseline separation of BVIXα from the BVIXδ and -β isomers, but no baseline separation between the BVIXδ and -β isomers  MTBLS3555.The authors acknowledge that the data presented in this study must be deposited and made publicly available in an acceptable repository, prior to publication. Frontiers cannot accept a manuscript that does not adhere to our open data policies. The data presented in the study are deposited in the MetaboLights repository, accession number ER and AW were involved in the initial concept and design of the experiment as well as the acquisition and analysis of the data. ER, AW, NF-D, and FX were involved in the subsequent experimental design and data interpretation, and the drafting and revising of the manuscript for publication. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Individual C60 molecules adsorbed on Cd(0001) may exhibit a bright or dim contrast in STM images. When deposited at low temperatures close to 100 K, C60 thin films present a curved structure to release strain due to dominant molecule–substrate interactions. Moreover, edge dislocation appears when two different wavy structures encounter each other, which has seldomly been observed in molecular self-assembly. When growth temperature rose, we found two forms of symmetric kagome lattice superstructures, 2 × 2 and 4 × 4, at room temperature (RT) and 310 K, respectively. The results provide new insight into the growth behavior of C60 films.We report an ultra-high vacuum low-temperature scanning tunneling microscopy (STM) study of the C M Au(111) ,34 and t60 monolayer, the individual C60 molecules appear as a bright or dim contrast [60 molecules combined with the multiple molecular orientations lead to the formation of a series of superstructures in the C60 monolayer, such as 2 × 2 [60 monolayers [60 thin film is under strain.Strain plays a central role in governing the structures of self-assembled thin films when organic molecules are deposited onto solid surfaces ,36,37,38contrast ,14,15,16as 2 × 2 ,26, 3 × as 2 × 2 , √19 × √as 2 × 2 ,29, 7 × as 2 × 2 . Moreovenolayers ,23. It iIn divalent metal Cd, there is a Coulomb repulsion between the 4d electrons and conduction band electrons, which leads to electrons with strong anisotropy in the mean free path and heat conduction. Recently, the Cd(0001) surface was shown to be a good substrate for observing and investigating strain phenomena of epitaxial films ,47.60-Cd system. The STM study demonstrated that the C60 thin films on the Cd(0001) surface presented diversity at different growth temperatures. When the growth temperature was close to 100 K, an unexpected wavy structure driven by compressive strain appeared. Two lines of the molecular arrangements were curved and corresponded to the √3 directions of the Cd(0001) surface. Moreover, an edge dislocation was observed when two neighboring wavy domains with different directions were encountered. When the growth temperature increased, two kinds of symmetric kagome lattice, superstructures (2 × 2 and 4 × 4) were identified in the C60 domains, where individual C60 molecules exhibited a bright or dim contrast.In this study, we used Cd(0001) thin films grown on Si(111)-7 × 7 as a substrate to explore the interface structures of the C60 molecules was deposited on the Cd(0001) surface at 100 K, they formed individual small clusters. 60 heptamer (seven-molecule cluster) with one central C60 molecule surrounded by six peripheral molecules. At the bias of 1.2 V, except for the central molecule with a dim protrusion, the other six peripheral C60 molecules revealed a similar bright contrast. When the bias was reduced to 0.5 V, the upper-right C60 molecule marked by the arrow became dim and revealed a two-lobe shape  direction present a wavy arrangement while the molecules in the  4 × 1 In surface [60 monolayer.When C surface . In addi60 films grown on Ge(100) [ Ge(100) . Unfortu60 row compared to Domain II. The same situation also occurs along the purple dotted lines. This observation indicates that the edge dislocation may occur in the monolayer regime and provides an intuitive STM diagram for the study of edge dislocations. Moreover, we noticed that this type of dislocation is absent when Cd(0001) substrate is kept at RT during C60 deposition.60 monolayer. 60 molecules adopt the same orientation. Each molecule reveals a two-lobe contrast corresponding to the C60 orientation with a 6:6 bond facing upward, similar to the C60 molecule in Au(111) and graphene [a1 = 10.0 ± 0.1 Å, close to the same value (10.02 Å) in C60 crystals [60 molecules adopt the same orientation. 60 molecule is surrounded by six bright molecules, constituting a head-to-head arrangement of hexagonal rings. In other words, the C60 monolayer is composed of two types of molecular rows: In row-b, all the molecules show a bright protrusion, while in row-a, one bright and one dim C60 molecule alternately appear. As a result, the bright and dim molecules constitute a 2 × 2 superstructure, with each unit cell containing four molecules. The measured intermolecular distance is a2 = 10.2 ± 0.1 Å, indicating a 2% tensile strain in the R33° domain. These types of symmetric kagome lattices also exist in the reported C60 domains in Au(111) [When growth temperature is raised to RT, two types of domain appear in the Cgraphene ,22. The crystals . This me Au(111) ,23.60 monolayer presents a larger kagome lattice with a 4 × 4 superstructure, shown in a3 = 10.5 ± 0.1 Å, much larger than the value (10.02 Å) in C60 crystals. The tensile strain is as large as 5%. These properties indicate that the packing density of C60 molecules decreases with an increase in growth temperature due to thermal expansion of the C60 layer. As displayed in 60 heptamer located at the inner hexagon and nine peripheral molecules. The heptamer consists of a central dim molecule and six bright surrounding molecules. We noticed that the C60 monolayer in this domain is composed of three types of molecular rows: all C60 molecules in row-a show a dim contrast; two dim molecules and two bright molecules are alternately arranged in row-b; one bright and one dim molecule appear alternately in row-c.When the growth temperature rises to 310 K, the C−10 Torr. A clean surface of Si(111)-7 × 7 was obtained through thermal flashing to 1550 K after uninterrupted degassing at 820 K for more than 7 hours. The flat and smooth Cd(0001) thin film was treated by depositing 15-20 monolayers onto the surface of Si-7 × 7. C60 molecules were evaporated from a homemade Tantalum boat at a rate of 0.4 ML/min onto the Cd(0001) thin films. After the process was completed, C60/Cd/Si was transferred into the STM observation chamber. The entire data were obtained in a constant current mode at 78 K (liquid nitrogen temperature).The experiments were performed in an ultra-high vacuum low-temperature scanning tunneling microscopy  with a base pressure close to 1.5 × 1060 molecules on Cd(0001) substrate in three different growth temperatures . STM studies demonstrated that C60 molecules are bright or dim in the thin film. At 100 K, we found the isolated C60 seven-molecule cluster, which indicates that the C60 heptamer can exist alone. As the molecule coverage increased, an unexpected wavy structure appeared, which suggests the C60 submonolayer was subjected to a large compressive strain. As a normal reaction, when under large external stress, the film of atoms or molecules may present larger bulk density or form superstructures. In this study, the arrangement of C60 curved instead of the aforementioned action. Additionally, an edge dislocation was captured when two different wavy structures encountered each other, which may develop a new phenomenon to study edge dislocation. Two types of symmetric kagome lattices were found at elevated growth temperatures, the 2 × 2 superstructure in the R33°domain with a 2% tensile strain at RT, and the 4 × 4 superstructure in the R44° domain with a 5% tensile strain at 310 K. Our results provide a new routine to fabricate and investigate pressure-resistant materials.In summary, our research showed the morphology of C


Ginkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition in HeLa-UGT1A1 cells (Hela cells overexpressed hUGT1A1). Further investigations demonstrated that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells. These biflavones potently inhibit hUGT1A1 in both human liver microsomes (HLM) and living cells, with the IC50 values ranging from 0.075 to 0.41 μM in living cells. Inhibition kinetic analyses and docking simulations suggested that four tested biflavones potently inhibit hUGT1A1-catalyzed NHPN-O-glucuronidation in HLM via a mixed inhibition manner, showing the Ki values ranging from 0.07 to 0.74 μM. Collectively, our findings uncover the key constituents in GBL responsible for hUGT1A1 inhibition and decipher their inhibitory mechanisms against hUGT1A1, which will be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.Human UDP-glucuronosyltransferase 1A1 (hUGT1A1) is one of the most essential phase II enzymes in humans. Dysfunction or strong inhibition of hUGT1A1 may result in hyperbilirubinaemia and clinically relevant drug/herb-drug interactions (DDIs/HDIs). Recently, a high-throughput fluorescence-based assay was constructed by us to find the compounds/herbal extracts with strong inhibition against intracellular hUGT1A1. Following screening of over one hundred of herbal products, the extract of  In adulractions   and herbal constituents (such as flavonoids and phenolic acids) have been reported (ely weak . The priGinkgo biloba leaves (GBL) displayed the most potent hUGT1A1 inhibition potency, with the inhibition rate exceeding 95% at the dose of 100 μg/ml. This finding encouraged us to further investigate the inhibition potentials of GBL and its major constituents against hUGT1A1 in various enzyme sources including HeLa-UGT1A1 cells and human liver microsomes, as well as to identify the key constituents in GBL responsible for hUGT1A1 inhibition. Furthermore, the inhibition potential and the inhibitory mechanisms of the newly identified naturally occurring hUGT1A1 inhibitors from GBL were also investigated by performing an array of inhibition kinetic assays and docking simulations. All these studies are very helpful for researchers to understand the interactions between the used GBL and human drug-metabolizing enzymes, and also provide the key data to assess potential risks or beneficial effects of the used GBL by inhibition of hUGT1A1.Recently, a high-throughput cell-based fluorescence assay was developed by using an artificial specific fluorescence substrate (termed N-butyl-4-(4-hydroxyphenyl)-1,8-naphthalimide (NHPN)) as the probe substrate and the hUGT1A1 overexpressed Hela cells (HeLa-UGT1A1 cells) as the enzyme sources . With thGinkgo biloba leaf extract (GBL) was obtained from Shanghai Sine Promod Pharmaceutical Co., Ltd. , and the chemical analysis of Ginkgo biloba leaf extract was analyzed by using ultra high performance liquid Chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric  (O-glucuronide (NHPNG) were chemically synthesized by one of the authors (Ping Wang) according to the previously reported scheme . UDPGA trisodium salt and polyethylene glycol hexadecyl ether (Brij 58) were purchased from Sigma-Aldrich . Cell culture medium and fetal bovine serum were obtained from Hylcone . Phosphate buffered saline , Millipore water and LC grade organic solvent  were ordered from Tedia company  and used for all experiments.The standard  States) . Other 1 States) . N-butyld scheme , with thO-glucuronidation activity of HeLa-UGT1A1 cells were determined by using NHPN as the probe substrate , equipped with a DGU-20A5R vacuum degasser, two LC-20ADXR pumps, a SIL-20ACXR autosampler and a CTO-20A column oven, was used to quantify NHPN and NHPNG. Chromatographic separation of analytes was carried out on a shim-pack Acchrom HC-C18 analytical column , while the column temperature was kept at 40°C. The flow rate was kept at 0.5 ml/min. The mobile phase consisted of acetonitrile (A) and water containing 0.2% formic acid (B). The gradient of the mobile phase was as follows: 0–0.5 min, 80–60% B; 0.5–3.5 min, 60–5% B; 3.5–5 min, 5% B; 5–7.5 min, equilibration with 80% B. The excitation wavelength and the emission wavelength were set at 370 and 520 nm, respectively for detection.2 in Modified Eagle’s Medium (MEM) containing 0.1% antibiotic-antimycoticmix, supplemented with 10% fetal bovine serum (FBS). HeLa-UGT1A1 cells were seeded in 96-well plates and cultured for 24 h. The cells were incubated in the medium containing different kinds of herbal products  or chemical constituents of GBL extract  for 60 min at 37°C under 5% CO2, and co-incubated with NHPN  for another 60 min. The reaction was terminated by adding an equal volume of ice-cold acetonitrile to each well, and then centrifuged at 20,000×g for 20 min at 4°C. After centrifugation, 5 μl aliquots of the supernatant was taken for LC-FD analysis. The validation data for this cell-based flourescence assay can be found in our previous publication  and the enzymes. Inhibitors  were serially diluted to the required concentration by DMSO. The final concentration of DMSO in the incubation mixture was less than 1%. When HLM was used as the enzyme source, HLM (10 μg/ml) and Brij 58  were pre-incubated for 20 min at 4°C to disrupt the ER membrane to remove the latency. A concentration of 5 μg/ml was used when recombinant UGT1A1 was the enzyme source. After pre-incubation for 3 min at 37°C, UDPGA was added to the incubation mixture to initiate the reaction. After 60 min incubation at 37°C, the reaction was terminated by adding 200 μl ice-cold acetonitrile, and the incubation mixture was kept on ice. After centrifugation at 20,000×g for 20 min at 4°C, 5 μl aliquots of the supernatant was taken for LC-FD analysis.NHPN was used for evaluating the inhibitory effects of GBL extract on HLM. A typical incubation mixture with a total volume of 200 μl consisted of 50 mM Tris-HCl (pH = 7.4), 5 mM MgCl50).The residual activity of hUGT1A1 was determined as follows: Residual activity (%) = (the fluorescence intensity of NHPNG generated in the incubations with inhibitor)/(the fluorescence intensity of NHPNG generated in the incubations without inhibitor) × 100%. A set of incubations with the same fluorescent substrate concentration and different inhibitor concentrations were performed to determine the half maximal inhibition concentration (ICKi) values. The incubation and analytical sample preparation were conducted as described previously. Goodness-of-fit parameter and Akaike’s Information Criterion (AIC) were employed to select the most appropriate inhibition kinetic modes from the following three inhibition modes including competitive inhibition A set of incubations by changing both the fluorescent substrate concentration and the inhibitor concentration were performed to determine the inhibition kinetic modes and the corresponding inhibition constant ; Ki is the inhibition constant that describes the affinity of the inhibitor towards hUGT1A1. The corresponding Ki value of each tested inhibitor is calculated by utilizing the slopes of second plot from Lineweaver-Burk plot versus inhibitor concentrations.Here, Docking simulations were used to explore the binding modes of each inhibitor into hUGT1A1 by using AutoDock Vina (1.1.2) . The 3D 50 and Ki values were determined by using GraphPad Prism 7.0 .All assays were conducted in triplicate. Data was expressed as mean ± SD. IC-O-glucuronidation in HeLa-UGT1A1 cells, with an apparent IC50 value as low as 12.41 μg/ml.First, 127 herbal products were collected and their inhibitory effects on hUGT1A1 were assayed in HeLa-UGT1A1 cells at the final concentration of 10 μg/ml. As shown in 50 values ranging from 1.38 μM (kaempferol) to 64.56 μM (myricetin)  to 0.41 μM (bilobetin)  . A totalricetin) . Notablylobetin) . The fivlobetin) , which c-O-glucuronidation in HLM, with the IC50 values ranging from 0.15 to 0.67 μM. Notably, the inhibition potentials of four tested bioflavones against hUGT1A1 in HLM are similar to that in Hela-UGT1A1 cells. However, the IC50 value of amentoflavone in HLM is much lower than that in living cells (0.41 vs. 3.29 μM)  and in Hela-UGT1A1 cells (12.41 μg/ml)   . For GBL1 μg/ml) . In addi0.67 μM) . The inhvia hydrophobic interactions  and a variety of clinical drugs  . InhibitGinkgo biloba is among the most widely consumed herbal supplements and phytopharmaceutical drugs used in the world. The primary active constituents of GBL are flavonoids and terpene lactones, which account for 26.8 and 9.8% of the tested GBL in the present study, respectively . It is worthy to be noted that, amentoflavone, also baring a biflavone structure, showed a relatively weak inhibition on hUGT1A1, with the IC50 value more than 10-fold higher than those of the other four biflavones. Although it only differs from the others in the number of the methoxy groups, a lower logP value (3.49), which indicates a poorer permeability compared to the other four with logP values of 5.23–5.38, could serve as an explanation for its decreased inhibition potency on hUGT1A1 observed in Hela-UGT1A1 cells. The much lower IC50 value in HLM (0.41 μM) than that in Hela-UGT1A1 cells (3.29 μM) for amentoflavone may further support the poor permeability of this compound  (ectively . Our curcompound . However1 μg/ml) .Cmax) of the specific four biflavones which were found to be the most potent hUGT1A1 inhibitors in the present study, the Ki values for these four compounds ranging from 0.15 to 0.67 μM were much higher than the measured plasma Cmax of their bioflavone analogs orally administrated in rats  in healthy volunteers , which may be a plausible explanation for the mixed-inhibition mode of these newly identified hUGT1A1 inhibitors. In conclusion, our findings revealed that the biflavones in GBL are potent hUGT1A1 inhibitors, which can be very helpful for guiding the rational use of GBL-related herbal products in clinical settings.In summary, with the help of a newly designed high-throughput cell-based fluorescence assay, the inhibitory potentials of a number of commonly used herbal medicines against hUGT1A1 were assayed in living cells. The results demonstrated that GBL extract used in the present study displayed the most potent hUGT1A1 inhibition potency, with the inhibition rate exceeding 95% at the dose of 100 μg/ml. Further investigations showed that four biflavones including bilobetin, isoginkgetin, sciadopitysin and ginkgetin, are key constituents responsible for hUGT1A1 inhibition in living cells, while the flavonoids in GBL contribute to a lesser extent for hUGT1A1 inhibition. Inhibition kinetic analyses suggested that the biflavones in GBL potently inhibit hUGT1A1-catalyzed NHPN-


Our results demonstrated that the substrate context exhibits a huge impact on the histone arginine methylation activity of PRMTs. Although all the tested PRMTs methylate multiple free histones individually, they show a preference for one particular histone substrate in the context of the histone octamer. We found that PRMT1, PRMT3, PRMT5, PRMT6, PRMT7, and PRMT8 preferentially methylate histone H4, whereas PRMT4/coactivator-associated arginine methyltransferase 1 prefers histone H3. Importantly, neither reconstituted nor cell-extracted mononucleosomes could be methylated by any PRMTs tested. Structural analysis suggested that the electrostatic interaction may play a mechanistic role in priming the substrates for methylation by PRMT enzymes. Taken together, this work expands our knowledge on the molecular mechanisms of PRMT substrate recognition and has important implications for understanding cellular dynamics and kinetics of histone arginine methylation in regulating gene transcription and other chromatin-templated processes.Histone arginine methylation is a key post-translational modification that mediates epigenetic events that activate or repress gene transcription. Protein arginine methyltransferases (PRMTs) are the driving force for the process of arginine methylation, and the core histone proteins have been shown to be substrates for most PRMT family members. However, previous reports of the enzymatic activities of PRMTs on histones in the context of nucleosomes seem contradictory. Moreover, what governs nucleosomal substrate recognition of different PRMT members is not understood. We sought to address this key biological question by examining how different macromolecular contexts where the core histones reside may regulate arginine methylation catalyzed by individual PRMT members ( NG-monomethylarginine (MMA), asymmetric NG, NG-dimethylarginine (ADMA), and symmetric NG, N’G-dimethylarginine (SDMA). PRMTs are classified based on the type of methyl marks they deposit: PRMT1, PRMT2, PRMT3, PRMT4 (coactivator-associated arginine methyltransferase 1 [CARM1]), PRMT6, and PRMT8 are type I enzymes generating MMA and ADMA; PRMT5 and PRMT9 are type II enzymes generating MMA and SDMA; and PRMT7 is a type III enzyme generating only MMA . PRMT7 methylates all the four core histones in the free protein form: H2A, H2B, H3, and H4 , there ., PRMT5 , 52, 53.., PRMT5 , catalyz., PRMT5 , 54, and., PRMT5 . Also, g., PRMT5 , we used14C) [14C]SAM, and methyl acceptor substrates such as individual recombinant human histones , histone octamer, mononucleosome, or a peptide with a sequence of the N-terminal tail of histone H4, H3, or H2A. Given that two copies of each histone exist in the histone octamer, we calculated the appropriate amount of histone octamer to achieve equal molar concentrations of histone in each reaction. Methylation reactions proceeded for 30 min (unless noted otherwise), quenched with 6× Tris–tricine gel loading buffer, and heated for 10 min at 95 °C. Proteins were resolved by 16% SDS-PAGE using a Tris–tricine buffer system, and then stained with Coomassie brilliant blue to visualize protein loading across lanes. Afterward, gels were dried for 2 h at 70 °C and then exposed to a storage phosphor screen (PerkinElmer) for 72 h in the dark, unless stated otherwise.We used a gel-based phosphorimaging methyltransferase assay to determine the substrate specificity of different PRMTs in histone methylation . The realane 9). To further understand the pattern of substrate specificity of PRMT1 on histones, we performed the methyltransferase assay with the H2A/H2B dimer, H3/H4 tetramer, histone H3.1 octamer, and histone H3.3 octamer. As seen in E–H, in the context of H2A/H2B dimer, PRMT1 strongly methylated H2A, which was similar to the methylation of free H2A (comparing lane 3 and lane 8). However, when the H3/H4 tetramer was added to the H2A/H2B dimer solution, H2A could not be methylated anymore and instead PRMT1 solely methylated H4 (lane 5). Therefore, the H2A methylation site is shielded by the H3/H4 tetramer, and only the H4 methylation site is accessible by PRMT1 in the octamer state, which explains why H2A is only methylated in the free H2A and the H2A/H2B dimer forms but not in the octamers. Overall, PRMT1 is capable of methylating the individual histones H2A and H4 proteins but exhibited a preference for histone H4 in the context of the histone octamer.It is well known that PRMT1 asymmetrically dimethylates histone H4 at the arginine-3 site (H4R3), and this event promotes transcriptional activation , 57, 58.2FYT) and full-length (residues 1–544) PRMT3 (A and B). When the substrate was the histone octamer, there was only a weak detection of arginine methylation on H4 . The full-length PRMT3 strongly methylated the individual histone H2A over H4, whereas H2B and H3.3 were not methylated (C and D). Unlike the truncated PRMT3, the full-length PRMT3 clearly methylated the histone octamer and preferred histone H4 in this macromolecular context . Also, strong arginine methylation of the Ac-H4(1–20) peptide was observed with only the full-length PRMT3 under these reaction conditions. Regardless of truncated or full-length PRMT3, in the context of nucleosome, no histones were observed to be methylated (lane 8 in A and C). These results demonstrate that PRMT3 methylates histones H2A and H4 in the free protein form but only methylates H4 in the octamer form, and the N-terminal domain of PRMT3 impacts histone arginine methylation.Thus far, there are a few reports that mention the use of histone peptides (only H4-based sequences) as a substrate of PRMT3 , 49, 59,4) PRMT3  and S3. 4) PRMT3 , A and Bon on H4 A, lane 7thylated , C and D context C, lane 7et al.  by PRMT5/MEP50 than PRMT5 alone, which affirms that MEP50 plays a role in modulating substrate specificity of PRMT5 .We performed the phosphorimaging assay to determine whether there were differences in the substrate specificity of PRMT5 alone leosomes  and S6. leosomes . Interesof PRMT5 , 64, 65.14C]SAM, and 1 μM of substrate); however, histone arginine methylation by PRMT6 was not observed (data not shown). To improve the signal-to-background noise, the incubation time for the dried gel with the phosphor screen was increased to 96 h. Also, the reaction time was increased to 3 h at 30 °C with 0.5 μM PRMT6, 5 μM [14C]SAM, and 1 μM of substrate. With these conditions, a faint higher molecular weight band was observed in nearly all the lanes with PRMT6, which was caused by PRMT6 automethylation . We applied the same exposure time and reaction conditions to PRMT7 that were used for PRMT6 (3-h reaction time and a 96-h exposure with the phosphor screen). We observed arginine methylation of the individual histones H2A, H2B, and H4 by PRMT7, albeit H2A was the most heavily methylated substrate . DespiteA, lane 3). Consistent with the previous results for all the other PRMTs we examined, PRMT8 did not appear to methylate the mononucleosome.Given PRMT8 shares the highest degree of structural homology with PRMT1 , one migFig. 8i.e., H2A for PRMT1, PRMT3, PRMT5, PRMT6, PRMT7, PRMT8 and H3.3 for PRMT4). For all the PRMTs examined, we did not observe any arginine methylation of the HeLa mononucleosomes (G)  of PRMT7, we only observed arginine methylation of H2A by PRMT7 without any additional radiolabeling of higher molecular weight proteins  , 70. Theproteins .Figure 9e.g., 12 mM NaCl). The negatively charged phosphate backbone of DNA can form electrostatic interactions with the positively charged histones, especially with the lysine-and arginine-rich N-terminal histone tails , although the salt concentration was low (13.4 mM NaCl with PRMT1 and 16.5 mM NaCl with PRMT5).After observing the absence of arginine methylation activity of PRMTs on the mononucleosomes, we wondered if this was due to the relatively low salt (NaCl) concentrations in the final reaction mixture  were detrimental to PRMT1 or PRMT5/MEP50 enzyme activity, we performed a control experiment with simply histone H4 protein at low (10 mM) and high (100 and 200 mM NaCl) concentrations . For boti.e., H4K5ac, H4K8ac, H4K12ac, and H4K16ac) may enable arginine methylation of mononucleosomes by the PRMTs. Especially, it has been reported that acetylation of the N-terminal H4 tail affects PRMT1 and PRMT5/MEP50-mediated H4R3 methylation (lanes 2 and 5), and the unmodified mononucleosome was used as a negative control (lanes 3 and 6). Based on the phosphorimaging results, there was no methylation of mononucleosomes observed, even when the histone H4 tail was present in the hyperacetylated form. We summarized the results of all the methylation experiments in Although arginine methylation of mononucleosomes by the PRMTs was not detected under all the experimental concentrations, it could be possible that hyperacetylation of the N-terminal tail of histone H4  or the absence of recombinant mononucleosomes (lanes 3 and 5). The result clearly showed that methylation of histone H4 by PRMT1 and PRMT5/MEP50 was abolished in the presence of the nucleosome.Because there was no detectable arginine methylation of nucleosomes by PRMT1 or PRMT5, we wondered if the presence of nucleosome would affect the methylation of free histones by PRMT1 and PRMT5/MEP50. To examine the influence of nucleosomes, we tested the methylation level of recombinant histone H4 in the presence . Under this condition, protein structures and protein–protein interactions are retained. On the native PAGE gel (lanes 1 and 3), likely owing to their negative charges in a running buffer of pH 8 . On the other hand, the recombinant H4 protein (lane 2) could not migrate onto the native gel because histone H4 was highly positively charged (pI 11.4). Lane 4 showed that once PRMT1 was mixed with H4, the PRMT1 protein band disappeared on this gel, ascertaining formation of PRMT1–H4 interactive complex. But, the migrations of PRMT1 and the nucleosome were not mutually affected, highlighting there was no significant binding between PRMT1 and the mononucleosome. Therefore, we concluded that the nucleosome inhibited PRMT1 activity by not binding the enzyme, but there might be some other possibilities.To understand the mechanism for the inhibitory effect of nucleosome on histone H4 methylation by PRMT1, we performed protein interaction assays using the native PAGE  have been reported to catalyze the formation of MMA, ADMA, and/or SDMA marks on the core histones . Nonetheless, even if PRMT1 indeed methylates histone H3, the labeling is significantly weaker in comparison to H4.Consistent with the earlier reports, PRMT1 and PRMT4 can methylate more than one histone individually, yet these enzymes prefer to methylate mainly histone H4 or histone H3, respectively, in the histone octamer . Even thet al.  used mouet al. , 42. Whaet al. , and yet  in this study because this was readily available, and our previous study examined how PTMs on histone H4 could regulate PRMT3 catalysis   was much higher on H2A than H4  and S6, Fig. 5y PRMT5) , 51, 52.et al.  among the PRMT family members . Yet unl% among tin vivo . It woulet al.  , which prevents the histones from being recognized and modified by the PRMT enzymes.The inability of the PRMTs in methylating nucleosomes deserves much mechanistic investigation. We examined the surface potential of PRMT1 and found that the inner ring region of the homodimer where the active site and the entrance of the arginyl peptide reside is highly acidic (charged) . With recharged) . The elecharged) . It can charged) , 84, 85. , abbreviated as [14C]SAM, was purchased from PerkinElmer. All other chemical reagents were purchased from Fisher Scientific, Oakwood Chemicals, Acros Organics, Sigma–Aldrich, Alfa Aesar, BDH, Research Products International Corp, Macron Fine Chemicals, Bio-Rad, EpiCypher, Inc, or J. T. Baker.PMSF, kanamycin, ampicillin, and IPTG were purchased from Gold Biotechnology. The Escherichia coli and purified by immobilized metal (nickel) chelation chromatography as previously described  vector), His-tag truncated PRMT3 , His-tag PRMT3 , His-tag PRMT6 , and His-tag PRMT8  were expressed from escribed . Human r1H NMR using 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt as an internal standard as previously described (1H NMR spectra and concentration adjustment factor.Peptides Ac-H4(1–20) (sequence Ac-SGRGKGGKGLGKGGAKRHRK-OH), Ac-H2A(1–21) (sequence Ac-SGRGKQGGKARAKAKTRSSRA-OH), and Ac-H3(1–20) (sequence Ac-ARTKQTARKSTGGKAPRKQL-OH) were synthesized using Fmoc solid-phase peptide synthesis and purified as previously described . Peptideescribed . See Supescribed  for 1H N14C]SAM was combined with 6 μl of peptide, 6 μl of an equimolar amount of histone, or 6 μl of reaction buffer before initiating the reaction with 18 μl of PRMT. The final concentrations of each component in the reactions were 1 μM of peptide/histone, 5 μM of [14C]SAM, and 0.05, 0.5, or 0.2 μM of PRMT. Reactions proceeded for 30 min at 30 °C, unless noted otherwise, and quenched with 6 μl of 6× Tris–tricine gel loading buffer  and pulse vortexed. All samples are heated for 10 min at 95 °C. About 30 μl of reaction sample was loaded into each well of a 16% SDS-PAGE gel. To vary the NaCl concentrations, a separate reaction buffer was prepared at different NaCl concentrations (851 mM–1.87 M NaCl) and diluted with the mononucleosomes or histones prior to adding radiolabeled SAM and initiating the reaction with PRMTs. We took into consideration the NaCl from the stock solutions of histones and mononucleosomes, PRMT storage buffers, and reaction buffer. As a molecular weight reference, 7 μl of the Precision Plus Protein Dual Color Standard  was added to lane 10. Proteins were resolved with 100 V for 10 min followed by 140 V for 75 min in a Tris–tricine running buffer . Gels were soaked in Coomassie staining solution  for 20 min and then soaked in destaining solution  for 40 min. Gels were dried in a Bio-Rad Gel Dryer (model 583) under vacuum for 2 h on gradient mode at 70 °C. An Amersham Biosciences phosphor screen was exposed to the dried gels in the dark for 72 h before scanning the screens with a GE Storm 865 Phosphor Imager. Gels that did not stain well during the initial 1 h process of staining and destaining were rehydrated after the phosphorimaging to restain the proteins.Peptides, histones, nucleosomes, and PRMTs were kept on ice and diluted in reaction buffer  to prepare working solutions. About 6 μl of [The authors confirm that the data supporting the findings of this study are available within the article and its supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.