The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors. The propensity for uptake via these mechanisms is dependent on the peptide sequence, choice of cargo, in vitro model and can be influenced by experimental factors, including incubation temperature and the presence or absence of serum in media7. In a number of CPP studies an intact actin cytoskeleton has been proposed to be required for cell internalisation and CPPs inside and outside of cells can modify the actin cytoskeleton to influence cellular processes including CPP entry11.Cell penetrating peptides (CPPs) are a group of short sequences typically containing 5–30 amino acids that have been extensively investigated as carriers for intracellular delivery of various cargos including genetic material, peptides, proteins and nanoparticles15. Classically macropincytosis is induced in response to growth factor activation such as epidermal growth factor (EGF) binding to the EGF receptor, initially leading to extensive actin-dependent ruffling on the plasma membrane. This induces a “gulping effect” manifest as an increased uptake of extracellular fluid16. Much of the information known regarding growth factor induced and actin dependent macropinocytosis comes from studies on high EGFR expressing A431 skin epithelia cells and their response to EGF18. Of interest are observations that some CPPs under defined experimental conditions may induce plasma membrane effects similar to that seen upon growth factor activation21 and in line with this that they promote the concomitant uptake of dextran, a well characterised marker of fluid phase endocytosis24. Dextran itself, in addition to being widely used as a fluid phase endocytic probe has been extensively investigated as a drug delivery vector25.One endocytic pathway that is absolutely reliant on actin is macropinocytosis. When activated this process has the capacity to form large plasma membrane derived intracellular vesicles termed macropinosomes28. Other natural compounds and synthetic products such as Latrunculin B (Lat B) and Jasplakinolide (JAS) have been identified or developed to target the actin directly or indirectly and to disrupt its organisation and function29. Very few studies have investigated the effects of these other actin disrupters on CPP uptake though it is generally recognised that actin disruption universally inhibits CPP entry.Tools used routinely to examine the roles of the actin cytoskeleton in various cellular processes, including endocytosis and CPP entry are pharmacological/chemical inhibitors. The most notable such agent is the fungal metabolite cytochalasin D (Cyt D) which disrupts actin polymerisation and is a well characterised inhibitor of various endocytic mechanismsHere we show that the effects of actin disruption on uptake of CPPs and dextran is cell type dependant and in A431 skin epithelia, in complete contrast to HeLa cells, leads to a dramatic increase in uptake of EGFP-R8 and dextran but inhibits the uptake of R8-Alexa488. Together the data indicate that actin organisation has very different influences on uptake of these octaarginine and fluid phase conjugates and that actin could be targeted to enhance cellular uptake of drug delivery vectors.2/37 °C incubator as a subconfluent monolayer. Cells were obtained from the ATCC and were routinely tested for mycoplasma.HeLa  cells (ATCC Code CCL-2) or A431  cells (ATCC Code CRL-1555) were maintained in growth Dulbecco’s Modified Eagle’s medium (D-MEM) supplemented with foetal bovine serum  and penicillin/streptomycin (100 units/ml and 100 μg/ml respectively), herein referred to as complete medium in a humidified 5% CO® 488 C5 maleimide salt  as previously described30. Labelling and conjugate mass (2126.5 Da) was confirmed by matrix-assisted laser-desorption ionization–time-of-flight spectrometry and quantified by UV spectrometry. All labelled peptides were resuspended to a stock concentration of 1 mM in autoclaved distilled water, aliquoted and frozen at −80 °C until needed.Peptide octaarginine (R8), extended with GC at its C-terminal (RRRRRRRR-GC), was synthesized by American Peptide Company  and labelled with Alexa Fluor6, and R8 and stop codon followed by EcoRI. The fragment was cleaved by NdeI and EcoRI and cloned into a pET3b (Novagen) derivative without N-terminal T7.Tag sequence to construct E. Coli expression vector pEV3b31 that has NdeI and EcoRI sites within the multi cloning site. Proteins were produced in E. coli BL21 (DE3) cells and assessed for purity and molecular weight as described in Supplementary Information and shown in Supplementary Fig. Plasmids encoding histidine tagged - EGFP-octaarginine, or - EGFP referred to here as EGFP-R8 or EGFP, were a kind gift from Professor Shiroh Futaki, University of Kyoto, Japan. To produce the plasmid the DNA fragment encoding EGFP was amplified from pEGFP-N1 (Clontech) using primers possessing the sequences for NdeI followed by His5 cells/dish and 1.0 × 106 cells/dish and maintained in 2.5 mL D-MEM containing 10%, 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were then allowed to adhere for 24 hr at 37 °C/5% CO2 to reach a confluence of ~80%. On the day of experimentation, they were washed with PBS and then incubated with 100 µl serum-free D-MEM (thus reducing well described serum binding effects32) containing either 2 µM CPP conjugate (R8-Alexa488 or EGFP-R8), 5 µg/ml transferrin-Alexa 647 (TF-647) or 0.1 mg/ml dextran (10 kDa)-Alexa 647 (Dex-647). The cells were then incubated for 1 hr (or 30 min for TF-647) at 37 °C/5% CO2 before washing 3x with PBS (TF-647 or Dex-647) or PBS 0.5 mg/mL heparin (R8-conjugates). They were then washed once with PBS, prior to the addition of 2 mL of imaging medium  and analysis by live cell confocal microscopy.HeLa or A431 cells were seeded on 35 mm MatTek dishes  at respective densities of 5 × 102. Endocytic probes  or R8-conjugates were then incubated with the cells in the absence (control) or presence of the inhibitors and further incubated at 37 °C/5% CO2 for times specified in figure legends prior to analysis by confocal microscopy. For pulse chase experiments, A431 cells were incubated with 0.1 mg/ml Dex-647 for 2 hr (pulse) followed by washing and a further 4 hr (chase) in fresh complete growth medium. EGFP-R8 (2 µM) was then added for 1 hr in the absence or presence of 10 µM Cyt D. EGFP-R8 and Dex-647 subcellular distribution was then analysed in live cells using confocal microscopy. For low temperature experiments, cells were preincubated with Cyt D as above and then placed on ice for 2 min prior to addition of ice cold EGFP-R8. The cells were then maintained under these conditions for 1 hr prior to analysis by confocal microscopy.Cells were seeded on MatTek dishes as previously described and were washed with PBS before preincubation at 37 °C with diluent control (DMSO) or actin inhibitors: 10 µM Cyt D for 15 min; 0.1–1 µM Lat B for 30 min; 0.2–4 µM JAS for 45 min; 50–100 µM Y27632 for 4 hr in serum-free D-MEM at 37 °C/5% COAnalysis was performed on a Leica SP5 confocal laser scanning microscope, equipped with laser lines: 405 Blue Diode (Excitation wavelength 405 nm for Hoechst 33342) Argon (Excitation wavelength 488 nm for Alexa 488 or EGFP), Helium Neon Laser 1 ) and Laser 2 (Excitation wavelength 633 nm for Alexa 647 conjugates). All images presented in this study were obtained using a HCX PL APO 63x 1.4 NA oil immersion objective with Leica Type F immersion oil. Parameters “Gain” and “Off-set” of the individual photomultiplier tubes were adjusted between experiments in order to obtain optimal image acquisition avoiding saturation. For multi-channel image acquisition, the channels were scanned in a sequential recording mode to avoid spectral cross-talk caused by overlapping excitation and/or emission spectra of fluorophores. Acquisition mode “XYZ” and image resolutions (pixels/image) of 512 × 512 or 1024 × 1024 were selected and pinhole size was set to 1 Airy Unit. Depending on acquisition requirements the scan speed was set as either 400 or 700 Hz and for single section images, the same optical section was scanned 3x to generate a line average image. Captured images were then analysed using ImageJ. In some cases (live cell imaging) the same cells were also imaged using Differential Imaging Contrast microscopy (DIC).6 cells/well) or A431 (1.0 × 106 cells/well) cells in 2.5 mL D-MEM containing 10% FBS were seeded on round borosilicate No.1.5 cover slips in 6 –well plates and allowed to adhere for 24 hr at 37 °C/5% CO2, to reach a confluence of ~80%. The cells were then treated with actin disrupters as described above, washed 3x with PBS and fixed for 15 min at room temperature with 3% PFA in PBS. Cells were then washed 3x with PBS and permeabilised with 0.2% Triton X-100 in PBS at room temperature for 5 min, washed 3x with PBS and stained for cell nuclei with 1.0 µg/ml Hoechst 33342 in PBS and for polymerised actin with 1.0 µg/ml Rh-P in PBS for 15 min at room temperature. The cells were finally washed 3x with PBS and once with distilled water, coverslips were mounted in DAKO mounting medium on glass microscope slides and imaged by confocal microscopy19.A recently described method was utilised giving information on actin architecture at different regions of the cells termed basal and cell body and apex (CBA). For actin staining, HeLa (0.5 × 102O) or actin inhibitors as previously described. They were then loaded with serum-free DMEM containing either 2 µM EGFP-R8, 5 µg/ml TF-647 or 0.1 mg/ml Dex-647 in the presence or absence of actin inhibitors for times stipulated in the figure legends. The plates were then placed on ice to prevent further uptake and washed 3x with either ice-cold PBS or 0.5 mg/ml heparin in PBS. To remove the plasma membrane bound TF, cells were washed 3x with ice-cold PBS followed by an incubation for 1 min in ice-cold acid wash 33. All cells were then washed twice with PBS at room temperature before trypsinisation at 37 °C for 5 min before placing the detached cells in centrifuge tubes and centrifuging at 4 °C for 3 min at 800 × g. The obtained cell suspension was washed 3x with ice-cold PBS and finally resuspended in 300 µl ice-cold PBS for measurement of cell-associated fluorescence  by flow cytometry on a BD FACSVerse utilising a 488 laser and 527/32 filter for EGFP analysis and a 633 nm laser and 660/10 filter for Alexa 647. Cells were gated using FSC-A/SSC-A to isolate single cell populations and analysed using FlowJo.A431 cells were seeded onto 12-well plates and cultured for 24 hr to be ~80% confluent on the day of experiment. Cells were washed twice with PBS and then incubated with diluent control  the drug caused the formation of small actin aggregates at both the basal and cell body and apex (CBA) sections of the cells  binds directly to filamentous actin and unlike Cyt D and Lat B induces stabilisation of the existing actin filaments thus inhibiting actin disassembly µM Fig. . This loConfocal microscopy dramatic increase in EGFP-R8 uptake in JAS treated cells Fig.  and the 45. A number of chemical inhibitors of the Rho-ROCK signalling cascade have been developed, including compound Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride]. This has been shown to inhibit the kinase activity of a number of ROCKs by binding to their catalytic sites47. Initially, 1 and 10 μM concentrations of Y27632 were investigated on actin localisation based on published observations showing actin deformities at these concentrations48. Supplementary Fig. Actin dynamics and regulation are achieved through the concerted action of actin regulatory proteins whose activities are finely modulated by protein kinases. These include Rho-associated coiled-coil forming serine/threonine kinases ROCK I and II that have been identified as important downstream effectors of RhoA through their interaction with its GTP-bound formIn CPP uptake experiments, Y27632 treated cells displayed scattered EGFP-R8 fluorescence in small vesicles in contrast to the vesicle aggregates seen in cells incubated with the other actin disrupting agents Figs ,4 and 8A49. This is based on studies utilising chemical macropinocytosis inhibitors including, and almost exclusively Cyt D for actin inhibition. It should however be noted that the involvement of actin activity has been shown for other endocytic pathways such as clathrin-mediated endocytosis (CME)50. As shown here and in several other studies, Cyt D at concentrations >5 µM, completely destroys actin organisation and, unsurprisingly, cell morphology is also compromised. A number of other natural compounds and synthetic products have been identified or developed to target the actin cytoskeleton directly or indirectly and to disrupt its arrangement/function. These include Lat B, JAS and Y27632 and these inhibitors have not been used to support Cyt D data for drug delivery endocytosis studies, including CPPs. To our knowledge this is the first study that has analysed CPP uptake with actin inhibitors employing different mechanisms of action.Since the discovery of CPPs, considerable efforts have been made to elucidate their internalisation mechanisms. Focusing mainly on arginine rich variants, macropinocytosis has been proposed to be a major contributing pathway for delivery of low molecular weight fluorophores or larger cargo by these peptides51 but correlative information regarding the uptake mechanism of the proteins relative to that of the peptides attached to fluorophores is lacking. EGFP-Tat uptake has been proposed to be mediated via macropinocytosis and caveolae52. Both Tat peptide, octaarginine,  and shorter number of arginine residues  have also been shown to deliver proteins into cells to mediate biological effects55.By employing high content analysis of the actin architecture using confocal microscopy and then flow cytometry we were able to correlate actin disorganisation with cellular uptake of EGFP-R8 as a model protein-CPP conjugate. EGFP has been shown to be delivered into cells by R8 and R957. This potential for the involvement of cortical actin was identified by discovering a rather unexpected and highly significant increase in the uptake of 10 and 40 KDa dextran in Cyt D treated A431 cells that was not observed with Alexa conjugates of transferrin, Tat and R833.In this study, HeLa cells reacted in a somewhat predictable manner in response to actin breakdown with Cyt D showing a reduction in uptake of R8-Alexa488 and dextran. In this cell line EGFP-R8 uptake was also inhibited by Cyt D treatment suggesting a similar mechanism of uptake. We have previously shown that cortical actin in A431 cells is particularly prominent and suggested that this may have a role in the uptake of dextran that, under normal conditions, enters cells via constitutive pinocytosis; also termed fluid phase endocytosis. This can be differentiated from stimulus induced macropinocytosisIn this study in we show that dextran and also EGFP-R8 uptake is significantly increased in A431 treated with Cyt D and contrasts to that observed for R8-Alexa488 (inhibition) and transferrin (no change). This strongly suggests that the process by which these two R8 conjugates interact with and/or enter cells is different. It should be noted that the structure of both conjugates differ in their cargo-CPP orientation and also the linkers utilised between them. We consider that it is highly unlikely that these factors mediate the differences in endocytic profiles of these conjugates. Due to the similarities we see in entry for dextran and EGFP-R8, we hypothesise that the differences are driven by the cargo and not the orientation of the CPP. But this warrants further study, noting that EGFP alone does not enter these cells at the concentrations used here, demonstrating a clear role for R8 that may be interacting differently with cells when attached to a fluorophore or protein. In none of the experiments performed did we observe clearly defined cytosolic EGFP fluorescence in Cyt D treated cells demonstrating that the effects of this drug maintained the localisation of the protein within a membrane bound compartment.30 and by performing pulse chase experiments we now show that the same is true for EGFP-R8, demonstrating that it is trafficked to the same degradative compartments. From these combined studies we also highlight that traffic to lysosomes of R8 attached to different cargo is also entirely dependent on functional actin.We have previously shown that R8 and Tat peptides as Alexa conjugates traffic rapidly by endocytosis to reach lysosomes57. In polarised cells, however, this effect was limited to dextran entering via the basolateral surface and the retained dextran was observed in large structures as we observe for EGFP-R8. It remains to be determined whether EGFP-R8 would also behave in a similar manner when introduced to different surfaces of polarised cells and whether this is influenced by any differences in cortical tension between these surfaces. The nature of these large structures is unknown but may be formed by tubulation of the plasma membrane that has been shown to be caused by Cyt D58. The plasma membrane tension of mammalian cells is coordinated and maintained through the actin network, and this profoundly influences endocytosis60. The reduced cortical tension resulting from actin disruption in MDCK basolateral surfaces57 and here in A431, cells may favour the formation of invaginations and tubulation to allow entry of macromolecules. This strongly supports studies showing an increased uptake of 70 KDa dextran and the folic acid receptor in Cyt D treated cells62. Both folic acid and dextran have been extensively utilised as, respectively, targeting and passive vectors for drug delivery63.Our findings prompted us to further study EGFP-R8 uptake in cells treated with inhibitors of actin that operate via different mechanisms and affect actin dynamics in different ways. Like Cyt D, at effective actin disrupting concentrations, JAS and Lat B also affected cell morphology and increased EGFP-R8 uptake that was again highly enriched in large structures in particular regions of the cells. Previous studies in non-polarised epithelial MDCK cells demonstrated that JAS treated cells had much higher levels of internalised and retained dextran compared with control cells64. In A431 cells the effects of Y27632 on the actin cytoskeleton was more subtle than that observed with the agents that directly targeted actin. No change in cell morphology was observed in treated cells but actin distribution was very different at high 100 µM concentrations. Effects of this agent in human trabecular meshwork eye cell actin has been shown to be extremely prominent at much lower concentrations, showing a loss of filamentous actin65. In contrast to results observed with direct actin inhibitors, we observed a slight but significant decrease in the uptake of EGFP-R8 in cells treated with Y27632. To our knowledge no CPP studies have been performed with this drug though it has previously been shown to affect the internalisation of plasma membrane receptors and also to inhibit (~50%) the uptake of 70 KDa dextran in macrophages68. This again supports our suggestion that dextran and EGFP-R8 uptake is responsive to the same inhibition in A431 cells but that they may not necessarily enter via the same mechanism. This supports recent observations showing that cell uptake profiles of HIV-TAT peptide and dextran can be separated69.Through interaction with the Rho GTPase, ROCKs play a central role in actin network assembly and membrane associationin vitro models obtained using actin modifying agents is difficult as the network that this protein nucleates is a regulator of so many processes; global cell morphology is often affected by its perturbation. Here we highlight that disruption of actin in some cell types promotes cell uptake of specific molecules whilst having no effects, or even inhibitory effects on others. From careful scrutiny of the literature there is precedent for this in other cell types and distinct plasma membrane regions in polarised cells. The possibility exists that these cells have a specific but common cortical organisation of actin that could be exploited to drive internalisation of drug delivery vectors carrying macromolecular therapeutics.Interpretation of endocytosis data in Supplementary Information


Electrochemical control over nitrogenase allows us to examine electrocatalytic proton reduction and potential-triggered CO inhibition using infrared spectroscopy. iii/ii) polyaminocarboxylate complexes as electron transfer mediators. This allows the potential dependence of proton reduction and inhibitor (CO) binding to the active site FeMo-cofactor to be established. Reduction of protons to H2 is catalyzed by the wild type MoFe protein and β-98Tyr→His and β-99Phe→His variants of the MoFe protein at potentials more negative than –800 mV (vs. SHE), with greater electrocatalytic proton reduction rates observed for the variants compared to the wild type protein. Electrocatalytic proton reduction is strongly attenuated by carbon monoxide (CO), and the potential-dependence of CO binding to the FeMo-cofactor is determined by in situ infrared (IR) spectroelectrochemistry. The vibrational wavenumbers for CO coordinated to the FeMo-cofactor are consistent with earlier IR studies on the MoFe protein with Fe protein/ATP as reductant showing that electrochemically generated states of the protein are closely related to states generated with the native Fe protein as electron donor.We demonstrate electrochemical control of the nitrogenase MoFe protein, in the absence of Fe protein or ATP, using europium( The molybdenum–iron (MoFe) protein (α2β2) houses the active site FeMo-cofactor [7Fe–9S–1Mo–C–homocitrate], FeMo-co, where substrates are reduced, and a [8Fe–7S] P cluster, that appears to function as an electron carrier. The Fe protein component (α2) contains a single [4Fe–4S] cluster and functions to deliver one electron to the MoFe protein during a transient association of the two proteins, in a process that is coupled to the hydrolysis of two equivalents of ATP for each electron transferred.1 The requirement for ATP hydrolysis by this system is energy intensive, comparable to the energy input required in the industrial Haber–Bosch process for N2 reduction where the energy comes primarily from fossil fuels. The complexity of the fully functioning nitrogenase system makes studies to define the mechanism of electron transfer and coupling with ATP challenging. The redox level of the MoFe protein is typically controlled indirectly by the ratio of the two component proteins  showed only minor structural changes that affected solvent ordering between the P cluster and FeMo-co, but no global structural changes.8 Here we show that it is possible to use the EuIII/II-ligand redox couples to mediate control by an electrode of wild type and amino acid substituted Azotobacter vinelandii nitrogenase MoFe protein, as evidenced by electrocatalytic proton reduction  contained within a layer of the polymer electrolyte, Nafion, titrated to pH 7.4 in Tris–HCl buffer  together with three ligands was added into the Nafion–Tris and the electrolyte to give Eu–BAPTA, Eu–EGTA and Eu–DTPA each at 0.25 mM .10 The same cell design was used for both electrochemical and attenuated total reflectance (ATR)-IR spectroelectrochemical experiments  are quoted relative to SHE via the conversion ESCE = –0.242 V vs. ESHE at 25 °C. To enable comparison between protein samples of slightly different concentrations, the currents are presented in units of μA (mg protein)–1. For MoFe protein lacking the active site FeMo-co  and the level of H2 detected in the headspace of the electrochemical cell, as measured by gas chromatography.Assignment of the electrochemical response at –900 mV as electrocatalytic proton reduction by the MoFe protein was confirmed in a separate experiment in a larger volume, stirred electrochemical cell –1 for wild type MoFe protein at –900 mV, representing approximately 1.4% of the rate observed in an assay with Fe protein and MgATP (2221 ± 66 nmol min–1 (mg protein)–1, see ESI Table S1II–EGTA or EuII–DTPA as electron donors.7 The fact that the low level of proton reduction by the apo-protein matches that of the control sample (‘no protein’) indicates that the FeMo-co centre within the wild type nitrogenase is responsible for its electrocatalytic proton reduction activity. Interestingly, the β-98Tyr→His and β-99Phe→His variants, which show significantly lower proton reduction activity than wild type nitrogenase in assays with the Fe protein as electron donor –DTPA to support the two-electron reduction of hydrazine by the β-98Tyr→His and β-99Phe→His variants, but not wild type MoFe protein.8These results demonstrate electrochemically-controlled substrate reduction by nitrogenase. The rate of electrocatalytic HN. Oxidation of this state to the ‘oxidised state’ termed MOX has a midpoint potential of –42 mV. The midpoint potentials for reduction of FeMo-co below the resting state are not well understood. Assuming that the Eu–L mediator system does not introduce any significant overpotential to catalysis by the MoFe protein, the onset of electrocatalytic proton reduction in the region of –800 to –900 mV suggests an approximate reductive driving force required for catalysis by nitrogenase. This is consistent with the estimated potential likely to be experienced by the MoFe protein during photocatalysis on CdS nanorods,6 although it represents a significant overpotential relative to the H+/H2 couple potential (E°′ = –437 mV at pH 7.4).At –300 mV, the most positive potential shown for the electrochemical experiments in 3 although inhibition of proton reduction by CO is weak and has only been detected at pH values above approximately pH 7.15 Proton reduction assays of wild type and the β-98Tyr→His and β-99Phe→His variants of the MoFe protein conducted at pH 7.4 with Fe protein as electron donor  to the MoFe protein. Under assay conditions using Fe protein/ATP, CO is known to inhibit reduction of all substrates catalysed by nitrogenase,Tyr→His variant is shown in Phe→His proteins. In electrochemical experiments conducted under a CO atmosphere, no hydrocarbon product of CO reduction was detected from either the wild type MoFe or variant MoFe proteins, showing that the proteins are not electrocatalytically reducing CO under these conditions.In electrochemical experiments on the MoFe protein with the Eu–L mediators at pH 7.4, CO is seen to inhibit proton reduction irreversibly at –900 mV, causing a substantial drop in the electrocatalytic current. A representative trace for the β-9818 and freeze-quench EPR and ENDOR.19 Several CO-bound species were observed in these studies, depending on the CO concentration. A crystal structure of the MoFe protein prepared under turnover conditions under a CO atmosphere showed a single enzyme form, in which the FeMo-cofactor had opened up with a bridging CO between two Fe sites on a face of the cluster.20Although proton reduction activity in the MoFe protein is generally reported to be fairly insensitive to CO in assays, CO-bound states of the MoFe protein prepared during turnover studies with Fe protein and ATP under a CO atmosphere have been detected by both stopped-flow IR spectroscopyνCO) at about 2143 cm–1, which typically shifts to lower wavenumber (ca. 2100–1800 cm–1) and becomes much more intense when CO coordinates to a metal centre, making it easy to determine features arising from CO ligands at metal sites.21 Potential-dependent difference spectra for the MoFe proteins, recorded under an atmosphere of 100% CO, are shown in νCO bands are observed at any of the applied potentials. The wild type MoFe protein and the β-98Tyr→His and the β-99Phe→His variants exhibit no νCO bands at the more positive potentials, but all show evidence of potential-induced coordination of CO at more negative potentials , in addition to several features at higher wavenumber (>2000 cm–1). The absence of any detectable νCO bands for the apo protein provides strong evidence that all the νCO bands observed for the wild type and β-98Tyr→His and β-99Phe→His variants of the MoFe protein arise from CO bound at FeMo-co rather than the P-cluster.We have applied ATR-IR spectroelectrochemistry to examine the effect of CO as a function of potential. Free CO in aqueous solution gives a weak IR stretching band  complex [CpMoCl(dppe)(CO)]+ has its νCO band at 2002 cm–1, while the Cp* derivative has νCO at 1971 cm–1.29‡Cp = cyclopentadienyl; Cp* = pentamethyl cyclopentadienyl; dppe = diphenyl phosphinoethane. The reduced Mo(ii) forms of these complexes have νCO bands at 1853 cm–1 and 1835 cm–1 respectively.29 The Mo(iii) complexes ((Me3SiNCH2CH2)3N)Mo(CO) and ((C6F5NCH2CH2)3N)Mo(CO) exhibit lower wavenumber νCO bands, at 1859 cm–1 and 1889 cm–1 respectively, however.30Additionally, a higher wavenumber band at 2050 cmνCO bands, consistent with the fact that electrocatalytic H+ reduction current does not recover when CO is flushed out with Ar –ligand mediated system, without the need for the Fe protein/ATP. The electrocatalytic rate of H2 evolution we measure is modest compared to the rate for H2 evolution with Fe protein/ATP as electron donor, or for photocatalytic H2 production by wild type MoFe protein on CdS (3000 nmol of H2 min–1 (mg protein)–1),6 but is greater than the rate observed in an assay with Eu–EGTA as electron donor without electrochemical regeneration (6.5 nmol of H2 min–1 (mg protein)–1).8The results presented here demonstrate that the nitrogenase MoFe protein can be placed under electrochemical control to drive substrate reduction or ligand binding using a Eu(Tyr→His and β-99Phe→His MoFe proteins using a combination of electrochemistry and infrared spectroelectrochemistry, showing the onset potential for H+ reduction and CO binding to be closely related. Several CO ligands appear to bind to the FeMo-co cluster, possibly at different redox levels, and give rise to IR νCO bands that closely resemble the potential-dependent features observed in earlier studies on isolated FeMo-co, as well as those observed for the MoFe protein under turnover conditions with Fe protein/ATP as reductant. This approach opens up opportunities for future work to examine, systematically, electron transfer and ligand binding reactions to nitrogenase MoFe protein without the complexities of the Fe protein/ATP as a reductant system.The Eu–L mediated electrocatalytic method was then used to examine the inhibitory effect of CO on electrocatalytic proton reduction by wild type and β-98


EF) with increasing temperature, as well as Pauli blocking caused by thermally excited electrons above EF. Our findings not only elucidate the mechanism governing the tunable Fano resonance but also open a route for exploring exotic physical phenomena through phonon properties in Weyl semimetals.Strong coupling between discrete phonon and continuous electron–hole pair excitations can induce a pronounced asymmetry in the phonon line shape, known as the Fano resonance. This effect has been observed in various systems. Here we reveal explicit evidence for strong coupling between an infrared-active phonon and electronic transitions near the Weyl points through the observation of a Fano resonance in the Weyl semimetal TaAs. The resulting asymmetry in the phonon line shape, conspicuous at low temperatures, diminishes continuously with increasing temperature. This behaviour originates from the suppression of electronic transitions near the Weyl points due to the decreasing occupation of electronic states below the Fermi level ( et al. observe a Fano resonance in the Weyl semimetal TaAs, revealing evidence for a strong coupling between phonons and Weyl fermions.The study of lattice vibrations coupled to electronic excitations may provide an avenue for exploring exotic physical phenomena. Here, Xu  EF in three-dimensional momentum space34567EF in these materials, interband electronic transitions near the Weyl points occur at a very low energy, 2|μ|, where |μ| represents the chemical potential with respect to the Weyl points9The Weyl semimetal (WSM) phase, a novel topological state of quantum matter, has been proposed to exist in materials with two non-degenerate bands crossing at Tc superconductors18q2, a parameter that describes the asymmetry of the Fano line shape, only reaching ∼0.04 , much greater than EF, making it impossible to tune the resonance via temperature1213This is actually a rare occurrence. Fano resonances generally do not occur in conventional metals or semiconductors, since the energy scale of interband electronic transitions in these materials is usually much higher than phonon excitations. However, this interesting phenomenon has been reported in bilayer or few-layer graphene121315.04 refs , 18, 19.Here we observe an intrinsic, strong High-quality single crystals of TaAs were synthesized through a chemical vapour transport methodR(ω) measured on the (107) surface of TaAs at 11 different temperatures from 5 to 300 K (ref. R(ω) that approaches unity at zero frequency is consistent with the metallic nature of TaAs. A well-defined plasma edge in the far-infrared region suggests very low carrier density, in agreement with the tiny volumes enclosed by the Fermi surfaces in this material345R(ω), a sharp feature can be clearly identified at ∼253 cm−1 (31 meV), as indicated by the arrow, which is associated with the infrared-active A1 phonon mode (d on the 7 surfacenon mode .σ1(ω) from R(ω) using a Kramers–Kronig analysisσ1(ω) of TaAs on the (107) surface in the far-infrared region at different temperatures. The low-frequency σ1(ω) is dominated by a narrow Drude response alongside prominent linear features, whose origin and temperature dependence have been previously discussed in detail1 mode manifests itself as a sharp peak in the σ1(ω) spectrum, as indicated by the arrow. σ1(ω) in the frequency region of 240–270 cm−1, where the A1 mode can be seen more clearly. It is well known that, in the absence of strong electron–phonon coupling, the phonon exhibits a symmetric line shape in σ1(ω) that can be described by a Lorentz oscillator, as schematically illustrated in σ1(ω), known as the Fano line shape13141 mode in TaAs exhibits a striking asymmetric line shape at low temperatures, which is an unequivocal signature of strong electron–phonon coupling. More interestingly, the asymmetry of the phonon line shape diminishes as the temperature rises, suggesting that the coupling-induced Fano resonance in TaAs can be tuned by temperature.In order to gain direct information about this mode, we calculated the optical conductivity s on the 7 surface1 mode, we extract the phonon line shape by subtracting a linear electronic background in a narrow frequency range at all measured temperatures, as shown in To quantify the temperature dependence of the AZ0 is the vacuum impedance; ω0, γ and Ω correspond to the resonance frequency, linewidth and strength of the phonon, respectively; q is a dimensionless parameter that describes the asymmetry of the Fano profile. A larger 1/q2 indicates more conspicuous asymmetry in the phonon line shape, while for 1/q2=0, the symmetric Lorentz line shape is fully recovered. The solid lines in where q2 as a function of temperature. While 1/q2 adopts a large value of 1.1 at 5 K . Thus electronic transitions at the frequency of the A1 mode (253 cm−1) do not occur near W2. This implies that the A1 mode is unlikely to be coupled to the electronic transitions near W2. However, electronic transitions near W1 set in at ω>4 meV (∼32 cm−1) and can therefore overlap with the frequency of the A1 mode, suggesting that this mode is coupled to the electronic transitions near W1.5 K refs , 14, 18,|μ| refs , 10, mak1 mode to its strong coupling with the electronic transitions near W1, we proceed to understand the temperature dependence of this mode. In WSMs, since the Weyl points are in close proximity to EF, electronic transitions near the Weyl points can be dramatically affected by thermal excitations, thus changing the line shape of the phonon that is coupled to these transitions. EF are fully occupied (blue), while the states above EF are empty (white). In this case, interband transitions at the energy of the A1 mode ħω0 are strong, as indicated by the thick arrows. As the temperature increases from 5 to 300 K, thermal excitations cause vacant states to appear below EF and electronic states above EF to be partially occupied. Electronic transitions at ħω0 are significantly suppressed  is the joint electron–hole pair density of states at the frequency of the A1 mode ω0 for a given temperature T; and μph and μe−h represent the optical matrix elements for phonon and electron–hole pair excitations, respectively. In this equation, we note that raising T mainly modifies De−h by thermally exciting electrons to the electronic states above EF and creating holes below EF. Near the Weyl points, the finite-temperature joint electron–hole pair density of states at ħω0 takes the formwhere ħω0. The red solid curve in q2 (blue solid circles) using 1 mode and the electronic transitions near W1, which are continuously suppressed with increasing temperature due to the reduced occupation of the electronic states below EF and Pauli blocking from thermally excited electrons above EF.where 1 phonon linewidth γ (26γph−ph(T) for this process followsA careful examination of the temperature dependence of the Aewidth γ  leads usγ0ph−ph is the residual linewidth at zero temperature. Apparently, this model does not account for the behaviour of the A1 mode in TaAs, since it gives an increasing γ as the temperature is raised, which is opposite to our experimental observation. Instead, strong electron–phonon coupling must be taken into account to understand the temperature dependence of γ in TaAs. In a system with strong electron–phonon coupling, a phonon can also decay by creating an electron–hole pairDe−h and is thus suppressed with increasing temperature due to thermal excitations, resulting in a temperature-dependent phonon linewidth γe−ph(T)where γ0e−ph represents a residual linewidth. Consequently, the temperature-dependent linewidth of a phonon mode that is strongly coupled to electronic excitations is given by γ(T)=γph−ph(T)+γe−ph(T). This equation gives an excellent description to the measured temperature dependence of the linewidth for the A1 mode in TaAs, as shown by the red solid curve in 1 mode is strongly coupled to the electronic transitions near W1, and both the line shape and linewidth of this phonon are closely tied to these transitions, which can be continuously tuned by temperature through varying the occupation of the electronic states near W1.where 1 mode  surface. Essentially identical behaviour was revealed for the A1 mode . We then1 mode , reachinħω0 are absent near W2 at low temperatures, Fermi smearing at high temperatures may relax the Pauli blocking, allowing these transitions to occur. From 1 mode arising from coupling to W2 at 300 K: 1 mode of TaAs over the measured temperature range (5–300 K). However, the enhancement of these transitions with increasing temperature may produce a strong Fano resonance at high enough temperatures. It is also worth pointing out that changing the occupation of the electronic states near W1 via other methods, such as electrical gating or femtosecond optical excitation, should induce a similar change in the Fano line shape of the A1 mode in TaAs.Finally, we note that although electronic transitions at E) and magnetic (B) fields pumps electrons from one Weyl point to the other with opposite chirality at a rate proportional to E·B, leading to a shift of EF in opposite directions at different Weyl points. This EF shift caused by the chiral anomaly can significantly affect the electronic transitions near the Weyl points, which accordingly changes the line shape of the phonon that is coupled to these transitions.The strong coupling between Weyl fermions and phonons enables the study of exotic quantum phenomena in WSMs by tracking the properties of phonons. One interesting proposal derived from our findings is to provide experimental evidence for the chiral anomaly212223EF shift, given by929For more insight, we can calculate the chiral anomaly-induced vF is the Fermi velocity and τ is the scattering time between different Weyl points. To obtain a realistic estimate, we take vF=5 × 105 m s−1  in TaAs through the observation of a Fano resonance. Varying the temperature, which changes the occupation of the electronic states in proximity to EF, can continuously tune the amplitude of the electronic transitions near W1, thus manipulating the line shape of the A1 mode. Our findings not only suggest a new approach for investigating exotic quantum phenomena in WSMs, such as the chiral anomaly, through the behaviour of phonons, but also imply a broad range of potential applications, such as in thermo-, magneto- and electro-optical devices. Please note that, after our manuscript was submitted, we noticed two preprints  as the transporting agent. After evacuating and sealing, the ampoule was kept at the growth temperature for 3 weeks. Large polyhedral crystals with dimensions up to 1.5 mm are obtained in a temperature field of ΔT=1,150–1,000 °C.High-quality TaAs single crystals were grown through a chemical vapour transport methodω) was measured over a broad frequency range on a Bruker VERTEX 80v FTIR spectrometer. In order to accurately measure the absolute R(ω) of the samples, an in situ gold overcoating techniqueω) from 40 to 15,000 cm−1 were measured at 11 different temperatures from 300 down to 5 K on three different surfaces of as-grown single crystals in an ARS-Helitran cryostat. Since a broad spectral range is required for a Kramers-Kronig analysis, we extended our data to the ultraviolet range  at room temperature using an AvaSpec-2048 × 14 fiber optic spectrometer.Near normal incident reflectivity R(σ1(ω) has been determined via a Kramers–Kronig analysis of R(ω)R=1−Aω−4) response.The real part of the optical conductivity All data that support the findings of this study are available from the corresponding authors upon request.How to cite this article: Xu, B. et al. Temperature-tunable Fano resonance induced by strong coupling between Weyl fermions and phonons in TaAs. Nat. Commun.8, 14933 doi: 10.1038/ncomms14933 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Note, Supplementary Figures and Supplementary References


The stability of a resulting toluene solution was also proven by static light scattering measurements over several days. The excellent solubility gives the possibility of an efficient and fast spray application of the functionalized graphene oxide quantum dots to steel surfaces. Hence, the macroscopic friction behavior was investigated with a Thwing-Albert FP-2250 friction tester. A thin film of the dodecyl amine functionalized graphene oxide quantum dots on steel lowered the friction coefficient from 0.17 to 0.11 and revealed a significant corrosion inhibition effect.Dodecyl amine edge functionalized few-layer graphene oxide quantum dots were synthesized in good yields. The covalent functionalization was demonstrated with NMR and AFM-IR. The resulting structure and particle size was measured with AFM and HRTEM. The thermal stability of the compound was investigated and showed a stability of up to 220 °C. The modified graphene oxide quantum dots showed excellent solubility in various organic solvents, including ethers, methanol, toluene,  Typically, lubricants are used to lower the frication of mechanical sliding contacts. Often the lubricant has also the task to prevent corrosion. It has been shown, that functionalized graphene and graphene oxide particles are able to inhibit corrosion4. Lubricants can be separated in liquid and solid lubricants. As liquid lubricants oils are commonly used while for solid lubricants graphite or molybdenum sulfite5 are applied. For many lubricants amines are the basic materials7. Recently, there have been reports that graphene and graphene oxide could be the base for solid and liquid lubricants11.Controlling the friction performance of mechanical contacts like bearings and gears minimize the energy cost and increases the lifetime of everyday mechanical components2-bonded carbon atoms13. It has attracted much attention in recent years due to its exceptional properties such as thermal and mechanical stability as well as electrical and optical activity19. Graphene with an average lateral dimension of 3–30 nm has been applied as graphene quantum dots in photoluminescence experiments24. Larger graphene sheets of up to 100 nm have been also classified as graphene quantum dots in this respect23. In other applications graphene sheets below 100 nm are defined as graphene nanosheets while graphene sheets larger than 100 nm are termed graphene microsheets25. If 2 to 5 graphene layers are dominating, the material has the term few-layer graphene while with up to 10 layers the name multi-layer graphene is used to define the material12. Graphene quantum dots have been so far mainly considered for sensing applications26. In addition, carbon quantum dots have found application in sensing, nanomedicine, photocatalysis and electrocatalysis27.Graphene is a two dimensional, single-atom-thick sheet of hexagonally constructed sp28. Furthermore, while graphene oxide often possess a C/O ratio of 4:1 reduced graphene oxide has often a ratio of 12:125. A further alternative to obtain graphene sheets is to stabilize and increase their solubility via chemical functionalization. The functionalization of graphene, graphene oxide and reduced graphene oxide can influence the behavior of these materials28. Particularly the solubility in different solvents and hence their processability can be increased via functionalization30. Especially the surface functionalization can increase the solubility and also stabilize single graphene sheets. However, this introduces sp3 centers on the graphene sheets, which can influence the behavior of graphene30. In consequence, the graphene sheets incorporate oxygen containing groups  which can be used to modify graphene oxide sheets with small organic molecules like octadecylamine (ODA)31, 5-(4-aminophenyl)-10,15,20-triphenyl porphyrin (TPP)32 or 2-(4-aminophenyl)ethanol33. The combination of amines with graphene oxide has shown a good performance in solid lubrification and additives for liquid lubrification35. The common technique for the assembly of the solid lubricants is a layer by layer assembly where the friction coefficient could change36. For example, a friction coefficient of 0.35 for a polymer vs. steel changed to 0.02 under nitrogen with a 300 nm thick polyethyleneimine/graphene oxide film. Under air a higher friction coefficient of 0.10 was observed34.A standard procedure to obtain graphene from graphite is to oxidize the latter to graphene oxide and transform it to reduced graphene oxide. However, reduced graphene oxide does not fully exhibit the behavior of graphene. Reduced graphene oxide sheets have defects in their sheet structure37 and by a modified Hummers method38. In addition, a modified procedure was reported by Haino et al. where graphite was treated with H2SO4 and HNO3 at 120 °C for 24 h which resulted in edge carboxylated graphene quantum dots with a diameter of ca. 21 nm39. Functionalization of the graphene quantum dots with benzyl amine derivatives resulted in material soluble in polar solvents like ethyl acetate or dichloromethane39. Hence, a functionalization or disruption of the surface of the graphene sheets was prevented39. Alternatively, graphene quantum dots can be isolated from coal40. Furthermore, graphene quantum dots were prepared from CX-72 carbon black in high yields and were applied successfully as probes for cellular imaging and have high potential in optoelectronic devices41. The utilization of carboxylic acid functions present in carbon nanoparticles provides a straightforward route to covalent functionalization with amines via the formation of an amide42. The reaction is easy to perform and the resulting amide functions are more resistant to hydrolysis compared to ester functions, since the NHR moiety is even a poorer leaving group than OR44.The behavior of graphene is also dependent on the size of the graphene sheets. If few- or multi-layer graphene is present in the material the solubility decreases. In addition, a decrease of the sheet size will aid to exfoliate graphene sheets. The most common approach to obtain graphene nanosheets from graphite is via electrochemical exfoliation3 centers are present in the sheets the more the original electronic behavior of the graphene nanosheets is preserved.Graphene quantum dots are considered to be the next generation carbon based nanomaterial. This is due to their outstanding physical, chemical and biological properties. In the present study we emphasized that the small size of the graphene quantum dot particles will ensure a good adsorption on the surface, especially with defect and fraction present on the natural surface of steel. In addition, since functionalized graphene oxide quantum dots have not been applied as lubricants so far, we were interested in the functionalization of graphene oxide quantum dots and to influence their behavior as lubricants for steel. In this process, a high solubility of graphene material in different solvents would be beneficial. The ability to predominantly functionalize the edges of graphene sheets leaves the sheet structure intact and the less spFluka AG, Merck AG, Lancaster, Alfa Aesar, Riedel de Haën and Sigma Aldrich. Unless specified otherwise the reagents were further purified by standard procedures. Graphite  and attenuated total reflection (ATR) spectra were measured with VERTEX 700 by Bruker. Raman spectra were recorded by InVia spectroscope in combination with DM2500 M-microscope from Leica Microsystems. GQDs 5 were dissolved in toluene to be dropped on a mica-wafer or carbon-pallets for atomic force microscopy (AFM) by Veeco Dimension Icon from Bruker or for scanning electron microscopy (SEM). To prove the functional groups on the GQDs all products were scanned by thermo gravimetric analyzer-mass (TGA-MS) spectroscopy TGA/SDTA851 by METTLER TOLEDO. X-ray diffraction (XRD) pattern was measured at room temperature using AXS D505 by Bruker. Tribological studies of steel flat samples against coated flat steel samples were performed with a Thwing-Albert FP-2250 Friction/Peel tester with a 2 kg force load cell. The sled has a contact area of 6 cm × 6 cm and a weight of 500 g. The distance between the sled and the load cell is 10.2 cm. The sled was placed on a clean steel surface and moved with a speed of 50 cm/min. The friction coefficient was calculated automatically. Transmission electron microscopy (TEM) was performed at 200 kV with a probe-side Cs-corrected TEM (JEOL JEM-ARM200F), equipped with a cold field emission gun, a post-column energy filter  and a JEOL energy dispersive X-ray spectroscopy (EDS) system. For this purpose, the toluene GOQs 5 suspensions (see below) were drop casted on a carbon coated Cu TEM grid. Even though it is known that at 200 kV graphene is destroyed by knock-on damage, we observed that the GQD were long enough stable in order to investigate them at this acceleration voltage. A similar observation was reported by Wilson and Sloan45.All reagents and solvents were purchased from commercial sources like COOH) 2 were synthesized in the presence of 65% sulfuric acid and 95% nitric acid (3:1)39 from Graphite ), purchased from Sigma Aldrich. The reaction mixture was treated with ultrasonic sound for 3 h to give a homogenous suspension. Afterwards the reaction was stirred at 120 °C for 1 d. The resulting product was neutralized with sodium hydroxide solution and washed with deionized water by use of ultrafiltration  to give GQDCOOH2.Oxidized graphene quantum dots (GQDCOOH2 (5 g) and oxalyl chloride . To slurry the reagents the reaction was treated with ultrasonic sound under reflux for 3 h. Subsequently the suspension was stirred for 3 d under reflux. The residual oxalyl chloride was removed by redestillation to give GQDCOCl3 (5.27 g) as grey powder, which was directly converted to the next step.Under inert gas conditions dry DMF (0.25 mL) was given slowly and dropwise to a reaction mixture of GQDCOCl3 (0.5 g), NEt3 (3 mL), amine 4  and DMAP  were added to dry DMF (10 mL) under nitrogen. The reaction mixture was stirred for 3 d under reflux at 110 °C under an N2-atmosphere. After completion of the reaction dichloromethane (5 mL) was added. The product was washed with acidulated (1 N HCl), bidestilled water (3 × 20 mL) and extracted with diethyl ether (3 × 30 mL) by using hydrophobic amine. Products functionalized with hydrophilic amine were purified by column chromatography using MeOH/CHCl3 as eluent. The degree of amide formation was determined by the content of nitrogen and oxygen by elementary analysis (w.%) , which corresponds to 80 mol%.GQD5 in toluene (0.1 g/L) was performed by using a commercial airbrush  supplied by N2 at 2.5 bar. The steel sheets were coated at a substrate-nozzle distance of 30 cm. Between two spray cycles the steel sheets were dried for 5 min at room temperature. The corrosion behavior was investigated using a steel sample with 20 spray cycles, which was emerged for 1 h in sea water (based on DIN 51358).Commercial Gardobond® cold-rolled steel sheets were cleaned by emerging in ethyl acetate. Before application, the steel sheets were dried for 1 h at room temperature. Thereafter, the spray deposition of the GQD measurements were performed with a Reference 600  potentiostat. A gold wire and a Ag/AgCl electrode were used as counter and reference electrodes, respectively. Impedance data were collected in artificial seawater for a frequency of 0.1 Hz by superimposing a 10 mV AC voltage at open circuit potential for 1 h.2O, 4 g sodium sulfate and 1 g calcium chloride were dissolved in 1 L of water. The mixture was thoroughly stirred and adjusted by dropwise addition of 0.1 N sodium carbonate (Na2CO3) solution.25 g sodium chloride, 11 g magnesium chloride*6 H39. Flaked graphite was treated with H2SO4 and HNO3 at 120 °C for 24 h as depicted in Fig. COOH2 was analyzed by XRD, Raman and AFM (see supporting information). In order to functionalize the carboxylic acid groups at the edges of graphene sheets, the carboxylic acid functions were first transferred into carboxylic acid chloride groups. Therefore, the material was treated with oxalyl chloride as shown in Fig. COCl3 was analyzed (see supporting information). Due to the moisture sensitive carboxylic chloride functions, GQDCOCl3 agglomerated on the AFM waver. Hence GQDCOCl3 was directly used in the subsequent step. The as-prepared material was treated directly with dodecyl amine 4 in order to form the desired carboxylic acid amides  were prepared according to a modified literature proceduredes Fig. . With do2, 3 and 5 see supporting information). The GQD dodecyl amine compound 5 showed a higher thermal resistance than pure dodecyl amine46. Due to the high solubility of the material in different solvents (for pictures of solutions with different solvents see supporting information) an 1H-NMR spectrum in deuterated toluene showed the formation of an amide group with a signal at 7.9 ppm in deuterated toluene  at 1020 cm−1 49, amide I at 1650 cm−1 49, amide II at 1540 cm−1 49, amide III at 1260 cm−1 ν (CH2), and a C=O stretch at 1720 cm−1. .For the identification of the sucessful amide formation an additional atomic force microscope infrared-spectroscopy measurment was performed Fig. . It show5 was investigated via HRTEM. An overview of the sample is shown in Fig. In addition, GQDs 2 hybridized carbon in graphene and smaller bond lengths reported for C=C bonds at lower hybridization50. The thickness of individual GQDs was determined by energy-filtered TEM using the log-ratio method and electron mean free path estimations according to the Malis model51 to be 2–3 nm.This is further supported by a HRTEM structure determination depicted in Fig. 5, the latter were dissolved in toluene and the resulting solution was observed for several days with static light scattering . The result is depicted in Fig. 5 coated steel is about an order of magnitude higher than that of uncoated steel. In both case the corrosion resistance slightly increases over time most probably based on the formation of corrosion products that slow down the interfacial transfer reactions. The impedance measurements thereby support the qualitative observations of the seawater immersion test52.Based on the time-dependent measurements of the impedance at 0.1 Hz Fig. , it gets5 applied as a lubricant can inhibit tribo-corrosion. The protecting mechanism is resulting from a barrier property of the dodecyl amine functionalized GQDs 5 film. Taking into consideration that the film thickness is at 20 spray cycles only 17 nm  the resulting effect is remarkable. Often a higher coating thickness is found with other carbon based coatings based on graphite10.Hence, 10. In addition, a better friction coefficient was observed compared to alkylated graphene oxide systems in combination with oil11. The presented results will help to further improve the development of tribo-corrosion inhibiting lubricant systems based on graphene derivatives.Graphene quantum dots (GQDs) and dodecyl amine functionalized GQDs have been prepared and investigated as lubricant. The resulting modified GQDs showed very good solubilities in numerous organic solvents, including ethers, methanol, n-hexane, heptane, xylene, dichloromethane and toluene. The good solubility provided the possibility of an efficient and fast spray application of the functionalized GQDs to a steel surface. In the presented work, it was possible to show with different methods that the successful covalent modification of GQDs with dodecyl amine was achieved. The resulting particles had an average size between 5 and 40 nm. The functionalized GQDs could be applied in spray coating to create a thin lubricant film on steel. The resulting lubricant film reduced the friction coefficient from 0.17 to 0.11 on the macro scale and it was shown for the first time that this graphene quantum dot based material has a significant corrosion inhibition effect. Compared to unfunctionalized graphene oxide, the functionalisation caused a significant enhancement of the lubricantSupplementary Information


Thaumatotibia leucotreta) by experimental manipulation of oxygen availability while measuring metabolic rate, critical thermal minimum (CTmin), supercooling point and changes in 43 metabolites in moth larvae at three key timepoints . Furthermore, we determined the critical oxygen partial pressure below which metabolic rate was suppressed (c. 4.5 kPa). Results showed that altering oxygen availability did not affect  CTmin nor  supercooling point. Metabolomic profiling revealed the upregulation of anaerobic metabolites and alterations in concentrations of citric acid cycle intermediates during and after chill coma exposure. Hypoxia exacerbated the anaerobic metabolite responses induced by low temperatures. These results suggest that cold tolerance of T. leucotreta larvae is not set by oxygen limitation, and that anaerobic metabolism in these larvae may contribute to their ability to survive in necrotic fruit.Insect cold tolerance depends on their ability to withstand or repair perturbations in cellular homeostasis caused by low temperature stress. Decreased oxygen availability (hypoxia) can interact with low temperature tolerance, often improving insect survival. One mechanism proposed for such responses is that whole-animal cold tolerance is set by a transition to anaerobic metabolism. Here, we provide a test of this hypothesis in an insect model system ( At temperatures below CTmin, insects enter an inactive coma like state, characterised by the absence of neurological activity  may interact with low temperature stress responses - affecting both benign and inducible forms of several traits associated with cold stress resistance - in at least some terrestrial insect species.The proposed mechanisms underlying insect chill coma can be grouped into three main categories: i) whole-organism oxygen limitation, ii) signal transmission failure and iii) disruption of ion regulation121314151819et al.Gryllus pennsylvanicus, while Michaud et al.Belgica antarctica exposed to −10 °C, suggesting a switch to anaerobic metabolic pathways. However, there is little consensus on the mechanisms at play during cold and hypoxia stressors. The role of OCLTT in setting low temperature limits in terrestrial insects has not been well examined to date as most studies have focused on high temperature responses, and then typically only examined whole-animal metabolic rates  ). Although the supercooling point (SCP) is not necessarily a useful measure of low temperature tolerance as its association with mortality depends on the species’ freeze tolerance strategy, in T. leucotreta larval SCP is equivalent to mortality temperaturesT. leucotreta are chill-susceptible with a CTmin of c. 6.7 °C when fed and a SCP of c. −15.6 °C under standard, benign laboratory rearing conditionsTo investigate the hypothesis that whole-animal oxygen limitation may set low temperature tolerance of insects we systematically investigated the influence of oxygen availability on several lethal and non-lethal metrics of low temperature tolerance. Here we make use of final instar larvae of a chill susceptiblemin under different controlled PO2. We estimated the critical oxygen partial pressure (Pcrit) for maintenance of whole-animal metabolic rate, and investigated changes in metabolites before, during and after chill coma. If chill coma endpoints are driven by oxygen availability, one major expectation is that hypoxia will increase CTmin (=less cold tolerant) while hyperoxia (increased oxygen) would decrease CTmin (=more cold tolerant)13O2 to assess if oxygen availability could influence this lethal estimate of low temperature tolerance. One prediction that can be made is that SCP would decrease under hypoxia if increased respiratory water loss, and the subsequent decrease in body water content, caused an increased concentration of solutes including cryoprotective molecules, while hyperoxia would be expected to have the opposite effect. In addition, OCLTT hypothesis predicts that if oxygen limitation is a primary driver of this functional low temperature performance limit, anaerobic metabolites should accumulate prior to CTmin under normoxia and hypoxia, but this effect should be relieved during hyperoxia. To examine this, 43 metabolites were measured across our suite of experimental conditions to test whether T. leucotreta likely employ anaerobic metabolism with the expectation that typical anaerobic metabolites such as lactic acid and alanine should be upregulated after hypoxic and potentially also low temperature exposures, but not hyperoxia. Furthermore, we predicted stronger anaerobic metabolite responses below, rather than above, Pcrit levels, with associated concomitant changes in low temperature tolerance more pronounced under conditions further from homeostasis setpoints.We determined the cold stress responses which are more frequently the focus of investigation in such studies by using thermolimit respirometrymin)O2 conditions . Using a flow-through respirometry setup, individual larvae were cooled from 15 °C to −15 °C at a cooling rate of 0.25 °C min−1. The rate of CO2 release (2) and activity data were analysed to determine CTmin (referred to as 2 CTmin and activity CTmin respectively) following methods outlined in Klok et al.et al.2 CTmin and activity CTmin were not significantly different from one another . The CTmin scored visually by an observer in a previous study2 CTmin at 21 kPa O2 , but was different from activity CTmin  = 0.001) . PO2 hadO2 were determined following a modified version of Boardman et al.T. leucotreta, was not affected by changes in PO2 .Supercooling points (SCP) under the six different Pcrit) following Basson and Terblanche2) was determined for fourteen larvae at six experimental oxygen concentrations . Pcrit was determined to be 4.5 kPa (2 (COV)O2 influenced spiracle behaviour as evidenced by 2 respirometry traces .Multiplexed respirometry was used to determine the critical oxygen partial pressure . Thereafter, insects were immediately returned to 15 °C and allowed 2 h to recover (“after chill coma”). The forty-three metabolites detected in T. leucotreta included 26 amino acids and peptides, 7 tricarboxylic (TCA) cycle metabolites, 9 other organic acids and a biogenic amine  at different timepoints before, during and after chill coma (P = 0.516). Variance of importance (VIP) scores indicated that component 1 was loaded by 2-ketoglutaric acid, lactic acid and margaric acid . As in the earlier results from “oxygen effects before chill coma”, changes in 2-ketoglutaric and lactic acids were the driving forces behind component 1 (47.7%) that separated the three groups, together with glutamine, malic acid, citric acid and alanine . 2-ketoglutaric acid remained the main contributor to component 1 (60.1%), followed by proline, histidine and citric acid. 2-ketoglutaric acid, proline and histidine were reduced under hypoxia, while citric acid was elevated under hypoxia  were significantly altered by oxygen availability during chill coma  or lethal (SCP) limits. This result is largely in keeping with the handful of other studies of tracheate arthropods that have investigated low temperature responses under altered oxygen levels13T. leucotreta appears capable of extracting sufficient oxygen to sustain aerobic metabolism from acutely hypoxic environments through careful regulation of their spiracles, excretory water loss and, by association, likely also ion regulation mechanisms. Finally, T. leucotreta clearly have some scope for anaerobic metabolism, which is likely to be of interest for post-harvest control of this pest when attempting to augment low temperature commodity disinfestation with modified atmospheres42T. leucotretaThree results of these experiments are most significant. First, we show here that O2 decreases, typically at values close to Pcrit, animals switch from being oxy-regulators to being oxy-conformers48Thaumatotibia leucotreta larvae likely follow this pattern, regulating normal processes above Pcrit, and switching to be oxy-conformers below Pcrit. The results of the metabolite profiling here further support this notion given that T. leucotreta larvae possess at least some ability to generate anaerobic metabolism end-products. This scope for anaerobic metabolism may be typical for larvae of holometabolous insects that routinely experience hypoxic conditions in artificial rearing mediums, necrotic fruit, or while wandering underground to pupateResponses of animals to hypoxia can be broadly classified as either the regulating class or the conforming class46crit of T. leucotreta to be c. 4.5 kPa, similar to other Lepidopteran larvae50O2 levelscrit is notoriously variable, depending on a suite of methodological factors3853crit ranged from 1.25 to 6 kPa O2 at 15 °C). One curious result, suggesting that larvae actively enhance gas exchange at rest under even lower levels of PO2, is that at 2.5 kPa, a value much lower than our estimated Pcrit, larvae likely employed active gas exchange to boost metabolism, or perhaps may be reflective of a second critical PO2 value showing the onset of anaerobic metabolism reported for marine organisms2 under hypoxia have also been documented in Drosophila at 3 kPa4056min at 2.5 kPa O2 are not significantly different to that estimated at 21 kPa O2, the nature of this increase in metabolic rate under hypoxia likely reflects a change in mode or pattern of gas exchange  in preparation for lower temperatures36O2 levels  in order to maintain cellular respiration, for example by keeping their spiracles open for longer to ensure sufficient oxygen supply to metabolically-active tissues1718192 levels . Regulatmin remained unaffected under the different experimental PO2 conditions. These results therefore do not match the general expectation that CTmin would increase under hypoxia and decrease during hyperoxia, but is a similar outcome to what has been reported in Tenebrio molitor beetlesO2. Therefore, the chill tolerance of T. leucotreta larvae are unlikely to be oxygen limited at low temperatures since exposure to PO2 did not alter their lower limits to activity (i.e. CTmin), nor the low temperature body freezing point (SCP). Our hypothesis regarding PO2 affecting SCP relied on significant changes in body water content. However, larvae survive up to 10 days without access to food and waterCTT. leucotreta larvae at all timepoints and treatments. This metabolite is an important intermediate in the Krebs cycle, and may also play a role as an antioxidant, much like histidine. Increased histidine has previously been associated with cold tolerance in Drosophila31The metabolomic profiling reported here revealed that 2-ketoglutaric acid was the main metabolite measured in Metabolomic results from within each oxygen treatment showed that lower temperatures under normoxia or experimentally-decreased oxygen treatments are likely associated with an increased reliance upon anaerobic metabolism, as indicated by elevated lactic acid . During minT. leucotreta low temperature toleranceWithin the timepoints measured , changes in oxygen availability have diverse effects. Before chill coma (at 15 °C), greater oxygen availability results in higher concentrations of amino acids and pyruvic acid, indicating higher rates of aerobic metabolism. see . During 6667The pathway analysis indicates that amino acid metabolism is significantly altered by oxygen availability during chill coma, both at translational and metabolic level . The patThaumatotibia leucotreta (Lepidoptera: Tortricidae) larvae were obtained from the Cedar Biocontrol Insectary, XSIT (Pty) Ltd, Citrusdal, South Africa and reared at 25 ± 5 °C at 50% relative humidity (L:D 12:12 h), under standard culture conditions False codling moth min) under different controlled PO2 conditionsO2  were obtained from compressed cylinders with balance nitrogen , while normoxic air (21 kPa O2) was generated using an aquarium pump. All air was passed through a set of scrubber columns containing soda lime and 50:50 silica gel:Drierite  to remove CO2 and H2O from the airstream. Rate of CO2 release (2) was measured in parts per million (ppm) using a calibrated Li-7000 infra-red gas analyser and data were recorded with standard LiCor software  on a desktop PC. Flow rate was maintained at 200 ml min−1 (STPD) using a mass flow control valve  connected to a mass flow control box  and activity was monitored electronically  in only a subset of individuals due to equipment constraints. Individual larvae were weighed before and after each respirometry run on a microbalance .Thermolimit respirometry (TLR)min program: hold at 15 °C for 30 min to allow larvae within respirometry cuvettes to equilibrate with bath temperature; cool down to −15 °C at a ramp rate of 0.25 °C min−1. Temperature logging with iButtons  revealed that the average cooling rate achieved by the bath during TLR recordings was ~0.24 °C min−1. This was repeated for n = 8 to 14 larvae for each PO2 . No individuals were re-used at another oxygen level.Individual larvae were recorded separately in 2 mL cuvettes, and cuvettes were submerged in a programmable circulating and refrigeration bath filled with ethanol  set to follow a CTO2 were determined following a modified version of Boardman et al.2), or a pressurised cylinder . The container was submerged in the programmable bath, set to follow the same temperature program as for TLR, except that cooling was continued down to −30 °C. Insect body temperatures were recorded at 1 Hz using a USB TC-08 thermocouple datalogger  connected to a desktop computer. The SCP was detected as the temperature just prior to the release of latent heat of crystallizationO2. SCP under 21 kPa O2 was repeated with both air directly from the aquarium pump and air scrubbed of CO2 and H2O (see respirometry setup above) in order to ensure no differences were obtained as a result of the source. Data were pooled as there was no significant difference between the scrubbed or unscrubbed air .Supercooling points under different Pcrit) following Basson and TerblancheMultiplexed respirometry was used to determine the critical oxygen partial pressure  was randomised. In all cases however, the final exposure to 0 kPa O2 was performed last as the effects of anoxia on these larvae are unknown and to avoid any potential damaging effects of reperfusion injury38−1 (~29 ml min−1 per channel) with the same gas as the active channel. All 14 individuals were therefore recorded at all six PO2 conditions. T. leucotreta larvae are able to survive at least 10 days at low (10%) relative humidity so the lengthy exposure to dry air was not considered to be problematicSeven larvae at a time were placed in individual 2 mL cuvettes maintained at 15 °C using the programmable bath . For each P2 was converted to μl h−1 using Expedata version 1.8.2 .All respirometry files (TLR and Pcrit experiments) were baseline drift-corrected and 2 and activity data were analysed to determine 2 CTmin and activity CTmin following methods similar to Klok et al.min usage in Stevens et al.2 data were converted to absolute difference sum . The actual inflection point of the ADS was calculated by selecting the period of data around the inflection point, and fitting a linear regression through this selection. The highest residual from this linear regression corresponds to the time at which 2 CTmin occurred. 2 CTmin (in °C) was calculated using the observed cooling rate of 0.24 °C min−1.For TLR, the min was calculated in a similar manner, with the exception that the data for the selection of the infection point was restricted to the portion of the file that corresponded to the end of activity . Activity data were not adequate for determining CTmin under anoxic conditions as the gas causes immobility of the larvae within 30 min (i.e. before cooling starts).Activity CT−1. In a small handful of files, certain variables were not able to calculated  and minimum metabolic rate (MetMin) during cooling (from 15 °C to 0 °C) were calculated. The temperature at which MetMin occurred (TMetMin) was calculated using the observed cooling rate of 0.24 °C mincrit, the central 20 min from each individual under each PO2 was targeted for analysis using custom written macros. In order to reduce the potential effects of activity on influencing Pcrit estimates, within this central 20 min, the mean of the most level 300 s was extracted to represent “resting metabolic rate” and the mean of the lowest 30 s was extracted to represent “minimum metabolic rate”. In order to measure the degree of spiracle activity, the coefficient of variation of 2 (COV) was calculated as the standard deviation divided by the mean 2O2.For PO2 as the categorical variable and body mass as the continuous predictor variable. All statistics were performed using Statistica software  unless otherwise mentioned. Overlapping 95% confidence intervals were used to identify homogenous groups.Data were checked for normality and equal variance, and where these assumptions were violated, non-parametric tests were used. Where body size (i.e. fresh mass) was a significant covariate, an analysis of covariance (ANCOVA) was used with P2 CTmin and activity CTmin were compared to data from Boardman et al.min was scored visually  using a non-parametric t-test. The 2 CTmin were compared between PO2 using an ANCOVA as mass was a significant correlate . Activity CTmin, MetAve, MetMin and TMetMin were compared using Kruskall-Wallis ANOVA. Differences in excretion events during cooling were compared using a generalized linear model with a binomial distribution and logit link function. Overall, start mass was significantly correlated with SCP  therefore an ANCOVA was used to compare the effects of different PO2 on SCP.The crit data can be analysed using a variety of common methods: ordinary least square regression, piece-wise linear regression, non-linear regression, t-tests and one-way repeated measures Friedman ANOVA3853crit using a boosted regression tree. Firstly, Wilcoxon matched pairs tests were used to compare between close PO2 groups  to identify the area in which the Pcrit lies. Secondly, an ordinary least-squares regression was fitted to the 2 data from 0, 2.5 and 5 kPa O2, and a second regression was fitted to the 2 data from 10, 21 and 40 kPa O2. The intercept of the two equations for these regressions was considered Pcrit. Lastly, 2 data were analysed using a boosted regression tree analysis with 2 as the dependent variable and PO2 as the continuous variable in Statistica v. 12. The three methods were applied on both resting and minimum metabolic rate data, and generally produced values in close agreement.PT. leucoptreta larvae under the different gas conditions and at different timepoints surrounding chill coma, we measured the change in metabolites following Koštál et al.2), a modified CTmin program was followed: temperature was held at 15 °C for 30 min (“before chill coma”); cooled down to −2 °C at a ramp rate of 0.25 °C min−1 and held at −2 °C for 30 min (“during chill coma”) immediately returned to 15 °C and allowed 2 h to recover (“after chill coma”). Samples were obtained at all 3 timepoints. All samples were immediately frozen in liquid nitrogen and stored at −80 °C. Prior to extraction, individuals were thawed and weighed on a microbalance  to determine fresh mass. Individual larvae were homogenised and metabolites were extracted in 70% ethanolIn order to investigate the metabolic changes in −1 and maintained for 2 min. Helium was used as the carrier gas and delivered at a constant flow rate of 1.1 mL min−1.The metabolomic profiles were extensively investigated by a combination of GC/MS and liquid chromatography (LC) coupled to MS (LC/MS) in the ethanolic extracts after their treatment with ethyl chloroformate under pyridine catalysis and simultaneous extraction in chloroform−1, using a gradient elution with the mobile phase consisting of (A) 5 mM ammonium formate in methanol and (B) 5 mM ammonium formate in water. The gradient elution program was linear from 30% to 100% A for 11 min, then held at 100% A for 1 min, and finally equilibrated for 4 min. The column eluent was directly introduced into a linear quadrupole ion trap mass spectrometer  equipped with a HESI II electrospray ionization source operated at 3.5 kV and scanning mass range 150–850 Da.LC/MS metabolite profiles were measured after evaporating a 30 μL aliquot of the chloroform extract to dryness by using a mild stream of nitrogen. After dissolution in 100 μL of the LC mobile phase, a 5 μL aliquot was injected into and separated on a Kinetex C18 column  at 35 °C at a flow rate of 400 μL min−1 using pooled sample mass. While there is a potential bias in scaling the data using fresh mass as body water content may have been altered by the PO2 treatments, data were analysed using a more conservative non-parametric GLZ using a normal distribution and identity link function.The data were processed with the Thermo Scientific Xcalibur 2.1 software and an in-house developed Metabolite Mapper platform, which provides automated peak detection and metabolite deconvolution by employing retention time and mass spectral and detector response features, followed by time alignment of the data obtained in each particular analysis for a defined experimental sample set and generation of data matrix, which is automatically exported to a predefined Microsoft Excel™ spreadsheet for further statistical processing. The metabolites were identified against relevant standards and further subjected to quantitative analysis by using an internal standard calibration method. All chemicals used were purchased from Sigma-Aldrich Co. , except the isotope-labeled metabolites used as internal calibration standards, which were obtained from Cambridge Isotope Laboratories . Whole-body concentrations of the metabolites were recalculated as nmol mgP-values were used to determine significance.For each of the 43 metabolites detected, the effects of oxygen and timepoint were analysed independently with a GLZ using a normal distribution and identity link function . Data were analysed both within timepoint to identify changes due solely to variation in oxygen levels and within an oxygen level to identify changes that are likely due to chill coma. Significance levels were adjusted for false discovery rate correction (FDR) using Benjamini-Hochberg procedureet al.Drosophila melanogaster specific library was conducted on the entire dataset for each timepoint independently to identify enriched metabolic pathways, and the impact of these enrichments. Globaltest pathway enrichment method was used, and the node importance measure was relative betweenness centrality.Multivariate analysis of whole-system metabolic changes among the oxygen treatments were conducted in MetaboAnalyst 2.0 and 3.076How to cite this article: Boardman, L. et al. Cold tolerance is unaffected by oxygen availability despite changes in anaerobic metabolism. Sci. Rep.6, 32856; doi: 10.1038/srep32856 (2016).


Saintpaulia ionantha is propagated by adventitious buds in horticulture, and periclinal chimeral cultivars are usually difficult to propagate. However, some periclinal chimeral cultivars can be propagated with adventitious buds, and the mechanism of which has been unknown. Striped flower cultivars “Kaname,” “Concord,” and “Monique” were used to investigate what causes flower color separation in adventitious shoot-derived plants by tissue culture. These cultivars were revealed to have mutated flavonoid 3′, 5′ hydroxylase (SiF3′5′H), WDR1 (SiWDR1), or flavonoid 3 hydroxylase (SiF3H), respectively, in their L1 layer. From our previous study using “Kaname,” all flowers from adventitious shoots were colored pink, which was the epidermal color of mother plants' flowers. We used “Concrd” and “Monique” from which we obtained not only monochromatic-colored plants the same as the epidermal color of mother plants, but also plants with a monochromatic colored plants, same as the subepidermal color, and a striped flower color the same as mother plants. Histological observations revealed that epidermal cells divided actively at 14 d after culture and they were involved in the formation of adventitious shoots in the cultured leaf segments of “Kaname.” On the other hand, in “Concord” and “Monique,” the number of divided cells in the subepidermis was rather higher than that of epidermal cells, and subepidermal cells were sometimes involved in shoot formation. In addition, the plant and leaf size of L1-derived plants from “Concord” and “Monique” were non-vigorous and smaller than those derived from the subepidermal layer. In conclusion, periclinal chimeral cultivars of Saintpaulia can be divided into two types. One type has a high cell division activity in the L1 layer, from which only single flower-colored plants derived from L1 can be obtained as adventitious shoots. Another type has a low cell division activity in the L1 layer, from which striped flower-colored plants the same as mother plants derived from several layers including L1 can be obtained as adventitious shoots. In the periclinal chimeral cultivar capable of propagation with adventitious shoots, the possibility was shown that cells in the L2 layer could form shoots by involving cells of the L1 layer with a low division activity. Saintpaulia ionantha, colloquially known as the African violet, adventitious shoots can be easily induced from most organs by tissue culture and therefore, it has been used as a model plant for studying adventitious shoot formation . Angiosperm SAMs are composed of several cell layers called L1, L2, and L3 that are numbered from the outermost layer  on their genome, while the inner layer had functional SiF3′5′H. All of their adventitious buds produced monochromatic pink flowers, indicating that only the epidermis layer contributed to adventitious bud ontogenesis in this cultivar , the pH of which was adjusted to 6–7 with dolomite. The light intensity of the greenhouse was controlled at around 250 μmol m−2 s−1 or less by covering with cheesecloths. The greenhouse temperature was maintained within 17°C–35°C by window ventilation and heating. Irrigation was carried out by water mist for 5 min at 9:00 a.m. at a rate of about 50 ml water per pot. Every other week, 50 ml of fertilizer  at 1/500 dilution was given while the temperature was adjusted to about 20°C to avoid a rapid leaf temperature drop which induces injury  from the three cultivars were used for the measurements. The statistical analyses used the multiple comparison test of Tukey-Kramer (P = 0.05) for “Concord,” and “Monique,” or a Student's t-test for “Kaname” (P = 0.05).Leaf blades of “Concord” and “Monique” mother plants were sterilized for 30 min with sodium hypochlorite (0.5% active chlorine) at around 20°C to avoid a rapid leaf temperature drop. Leaves were rinsed three times with 20°C sterile distilled water and cut into 1 × 1 cm squares with a sterile razor blade. They were plated on modified Murashige and Skoog (MS)  medium cTo observe cell division and SAM formation on the cultured leaf segments of “Kaname,” “Concord,” and “Monique,” the cultured samples were fixed in a formalin-acetic acid-alcohol (FAA) solution  after 0–18 d of culture and evacuated for several days in FAA. Then, the samples were washed with tap water and dehydrated through a series of different ethanol concentrations. Ethanol in the samples was replaced by a resin solution  and then embedded in Technovit 7100 at 25°C according to the manufacturer's instructions. The samples were cut into 5 μm-thick sections using a microtome  and the sections were placed onto glass slides. Sections were then stained with 0.05% (w/v) toluidine blue and observed under a VHX digital microscope . The number of cells and total cell number were counted in each epidermal layer and subepidermal layer within seven 2 × 2 mm randomly selected sections 0–14 d after culture for the three selected samples.Saintpaulia pigments were used as anthocyanin standards  to extract the pigments. HPLC analysis was performed using an LC10A system  with a C18 column  maintained at 40°C and a photodiode array detector. The detection wavelength was 530 and 350 nm for anthocyanins and flavones, respectively. Elutant A was 1.5% phosphate dissolved in water and elutant B was 1.5% phosphate, 20% acetic acid, and 25% acetonitrile dissolved in water. The analysis was performed at a flow rate of 1 ml/min and a column temperature of 40°C using a mobile phase gradient starting at 20–85% B over 40 min with 5 min re-equilibration at 20% B. Nine purified 1H spectra and 100 MHz for 13C spectra in dimethyl sulfoxide (DMSO)-d6. Chemical shifts are reported relative to DMSO (2.5 ppm) and coupling constants (J) are in Hz.UV-vis spectra for purified flavone was recorded on a UV-vis multi-purpose spectrophotometer  operated as a double-beam instrument in MeOH (from 200 to 500 nm). HR-FABMS were obtained in a positive ion mode using a 1:1 mixture of dithiothreitol and 3-nitrobenzyl alcohol as a matrix with the JMS-700 MS station mass spectrometer . NMR spectra were recorded on a JNM AL-400 spectrometer (JEOL Ltd.) at 400 MHz for 20 primer and ReverTra Ace  according to the manufacture's instruction. RNA concentrations were adjusted to 100 ng/μl by spectrophotometer . In “Concord,” the expression analysis was performed using RNA extracted from petals and leaves of white and blue monochromatic color plants  expression in “Monique,” the petals and leaves of monochromatic plants  and subjected to reverse transcription (RT) using an oligo-(dT) Figures . In “Mon Figures  were useSiCHS-A (DQ788862): chalcone synthase-A, SiF3H (LC269962), SiDFR (LC269960): dihydroflavonol 4-reductase, SiANS (LC269961): anthocyanidin synthase, as genes coding enzymes on the anthocyanin biosynthesis pathway and SiDEL (LC269959): DELILA/JAF13 (a basic helix-loop-helix), SiMyb2 (LC269957), SiWDR1 (LC269958), and SiPAC (LC269956) as transcriptional factors for anthocyanin biosynthetic genes were amplified by specific primers (Table SiActin (AB596843) was used as the reference gene.For the expressions of rs Table  which wetaq (TOYOBO), and 0.2 μM of forward and reverse primers at a final volume of 10 μl according to the manufacture's instruction. The program was set at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 55–60°C for 30 s depending on the value of the melting temperature of each primer set, and 72°C for 10–60 s depending on the length to be amplified. All PCR products were electrophoresed in 1% agarose gel and signals were detected under a UV-illuminator.For DNA extraction, the DNeasy Plant Mini Kit (QIAGEN) was used according to the manufactures' instructions. Moreover, in order to extract DNA from the L1 layer, trichomes were collected by a razor blade under a stereomicroscope and the DNA was extracted by mixing with 2 ml of buffer AP1 (QIAGEN) in plastic tubes and extracted according to the manufactures' instructions. Also, the epidermis-removed petiole was used as a sample in order to obtain DNA from internal layers. DNA concentrations were adjusted below 2 ng/μl by spectrophotometer . All PCR/RT-PCR mixtures contained 1 μl of template DNA (at a maximum 1–2 ng), 0.1 μl of Blend SiWDR1 in blue and white “Concord” mutants was determined by a commercial kit  and inverse PCR. According to the manufactures' instructions, genomic DNA  of “Concord” leaves of monochromatic blue flowers was digested by Xba I  and ligated with adaptors supplied by the kit. The first PCR was conducted with WDR40 Walk R1st primer (Table EcoRI (TaKaRa Bio) and self-ligated with 80 units of T4 DNA ligase . The ligated DNA was precipitated with ammonium acetate and ethanol and used for PCR. For the first PCR, 1st Primer-F aroundA and 1st Primer-R aroundA were used and for the nested PCR, B-F and 2nd Primer-R aroundA were used  was used according to the manufactures' instructions and a 5′-Full RACE Core set (TaKaRa Bio). The amplified products were cloned and sequenced as mentioned before. From the sequence data (LC2269962), primers for the total length amplification were designed and used for RT-PCR and genomic PCR using RNA/DNA of monochromatic blue flower plants. From these sequence data, the exon and intron structure of DNA of monochromatic blue flower plants was determined.To determine full length mRNA sequences of Flowers of adventitious shoots from leaf laminas of “Concord” and “Monique” exhibited three types of flowers. In “Concord” white monochromatic-colored plants (L1 phenotype) Figure  were obsSeven and nine anthocyanin derivatives were detected from “Concord” and “Monique” petals, respectively Table  by HPLC SiCHS-A, SiF3H, SiDFR, and SiANS in “Concord” petals were analyzed and the results showed that expressions of these genes, especially SiF3H, SiDFR, and SiANS, were lower in monochromatic white mutants compared to monochromatic blue mutants , and this product was not petal-specific because the direct sequence of RT-PCR product contained the nine SNPs sequences in addition to the SiWDR1 sequence (data not shown). Suppression of these enzymatic genes, including SiANS which is after the SiDFR step, was consistent with the taxifolin feeding experimental result. SiWDR1 plays the most important role for anthocyanin biosynthesis by complexing with other transcriptional factors and by regulating the expression of several enzymatic genes on the anthocyanin biosynthesis pathway , was connected to an unknown sequence  had disappeared from white mutant cells  from the SiF3′5′H promoter, and the blue color was determined to be a malvidin derivative and the pink pigment a pelargonidin derivative. During shoot regeneration, TE was transposed from the promoter region and many monochromatic blue mutants and periclinal mutants were obtained. Some of the periclinal chimera was L1 pink and L2 blue, which is the same pattern as “Kaname,” and the others were L1 blue and L2 pink which is the opposite color pattern to “Kaname.” So, this means that a periclinal chimera structure with a mutated SiF3′5′H L2 is possible. If the vigorous L1 covered the non-vigorous inner layer, it would be difficult for this type of periclinal chimera to maintain its structure. We often find some leaf variegated plants whose L1 are an unvigorous albino genotype, and which have been maintained for many years in vegetative propagation. This suggests that when the vigorous L2 is covered by weak L1, it makes the chimeral meristem structure stable. We can suppose that L1 cells are much vigorous than L2 cells. L1 cells will invade into the L2 and break the chimera structure. So, if the cells with a different vigor compose a SAM, the low-vigor cells will distribute at the outer cell layer, L1.In Two possibilities can be considered in the process of shoot regeneration of a periclinal chimera. One is a case where the L2 layer develops SAMs that include cells of the L1 layer, and the other is a case where a mosaic SAM with L1 and L2 cells occur and more stable periclinal structured SAMs are screened Figure . Here, wSaintpaulia cultivars, whose patterns are derived from a periclinal chimera structure, can be divided into two types. One type is cultivars whose regenerated shoots are derived from the epidermal layer, such as “Kaname,” whose epidermis undergoes active cell division during tissue culturing. Another type is cultivars that have a relatively high percentage of regenerated adventitious shoots that strictly adhere to their mothers' original chimeral structures, such as “Concord” and “Monique,” whose inner cells but not epidermal cells undergo active cell division during tissue culturing. The latter type of cultivars may be recognized by comparing growth characteristics of separated epidermis-derived plants and inner-derived plants, which show vigorous and less vigorous growth, respectively.In conclusion, we propose that the striped TN contributed to data collection, data analysis, and writing the draft manuscript; SY, SO, AD, and MD contributed to data analysis, data interpretation, drafting the work, and final approval of the version; KH and FT contributed to data collection, data analysis, data interpretation, drafting the work, and final approval of the version; MH contributed to the conception or design of the work, data analysis, data interpretation, writing the draft manuscript, and final approval of the 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.